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515284 | Site-specific mutagenesis of Drosophila proliferating cell nuclear antigen enhances its effects on calf thymus DNA polymerase δ | Background We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA) on DNA synthesis catalyzed by mammalian DNA polymerase δ(pol δ). In the presence of homologous PCNA, pol δ exhibits 1) increased absolute activity; 2) increased processivity of DNA synthesis; 3) stable binding of synthetic oligonucleotide template-primers (t 1/2 of the pol δ•PCNA•template-primer complex ≥2.5 h); and 4) enhanced synthesis of DNA opposite and beyond template base lesions. This last effect is potentially mutagenic in vivo . Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes. Results Drosophila PCNA, although highly similar in structure to mammalian PCNA (e.g., it is >70% identical to human PCNA in amino acid sequence), can only substitute poorly for either calf thymus or human PCNA (~10% as well) in affecting calf thymus pol δ. However, by mutating one or only a few amino acids in the region of Drosophila PCNA thought to interact with pol δ, all four effects can be enhanced dramatically. Conclusions Our results therefore suggest that all four above effects depend at least in part on the PCNA-pol δ interaction. Moreover unlike mammals, Drosophila offers the potential for immediate in vivo genetic analyses. Although it has proven difficult to obtain sufficient amounts of homologous pol δ for parallel in vitro biochemical studies, by altering Drosophila PCNA using site-directed mutagenesis as suggested by our results, in vitro biochemical studies may now be performed using human and/or calf thymus pol δ preparations. | Background Many Drosophila melanogaster homologs of the proteins required for both DNA replication and repair have been identified and in several cases purified to apparent homogeneity. These include DNA polymerase α holoenzyme [ 1 , 2 ], DNA polymerase δ(pol δ) [ 2 - 4 ], replication protein A (RP-A; [ 5 ]), replication factor C (RF-C; e.g., see [ 6 - 9 ]) and various origin recognition complex (ORC) subunits (see e.g., [ 10 , 11 ]). Moreover, complete replication of DNA containing the SV40 origin of replication has been reconstituted in vitro using purified SV40 T-antigen and Drosophila cell-free extracts [ 7 ]. A protein about which much information has been obtained is proliferating cell nuclear antigen (PCNA). Drosophila PCNA was first identified both as a highly purified protein able to substitute, albeit poorly, for human PCNA in a cell-free SV40 DNA replication system reconstituted from purified proteins [ 12 ] and by Yamaguchi et al. [ 13 ] who used an oligonucleotide probe to detect the Drosophila PCNA cDNA and gene, express the protein in E. coli and deduce its complete amino acid sequence. Further results indicated that in flies, PCNA was encoded by a single gene located at position 56F5-15 on the right arm of chromosome 2. This was subsequently identified as the Drosophila mus 209 locus [ 14 ]. Recently, a second Drosophila PCNA gene of limited homology to the original and of unknown biological function has also been found [ 15 ]. Protocols have been established for purification of wild-type human PCNA from tissue culture cells [ 16 , 17 ], unmodified wild-type human PCNA after regulated expression in E. coli [ 18 ] and NH 2 -terminally his-tagged but otherwise wild-type human PCNA, also engineered for bacterial expression [ 19 ]. All were comparably effective at stimulating mammalian pol δ. Similar protocols have been developed for Drosophila PCNA and strategies for site-directed mutagenesis have been devised and implemented [ 20 ]. Recently, Zhang et al. [ 21 ] (see also [ 22 ]) as well as others (e.g., see [ 23 ]) identified the interdomain connector loop of PCNA (amino acids 119-133 of human PCNA) as crucial for binding pol δ. Of note, relative to wild-type PCNA, mutations of the molecule within this region such as glutamine at position 125 changed to glutamic acid (Q125E) promoted increased pol δ-processivity [ 21 ]. In human PCNA, residues 123, 126, 127 and 128 were defined as being essential for interaction with pol δ [ 21 ]. Comparison of human with Drosophila PCNA sequences in this region indicated that of these four amino acids, three (residues 126, 127 and 128) are identical. The fourth, residue 123, is glutamine (Q123) in wild-type Drosophila PCNA. The corresponding residue in human PCNA is valine (V). To investigate the role of the interdomain connector loop of PCNA on the effects of PCNA on pol δ, we mutagenized residues within this region of Drosophila PCNA so that they more nearly resembled human amino acids. After bacterial expression and purification, we tested the effects of these site-specifically modified ("humanized") Drosophila PCNA molecules on purified calf thymus pol δ (two-subunit form; see [ 17 , 24 ]). Calf thymus and human pol δ are highly similar in amino acid sequence [ 25 - 27 ] and can, for our purposes, be used interchangeably. "Humanization" of a single Drosophila PCNA residue, conversion of Q123 to V (Q123V), conferred upon it, enhanced ability to affect several properties of calf thymus pol δ. More extensive mutagenesis, in which the entire interdomain connector loop of Drosophila PCNA (amino acids 119-133) was replaced by the corresponding human residues, was still more effective at stimulation of calf thymus pol δ, than either wild-type or Q123V Drosophila PCNA. However, it was considerably less effective than wild-type human PCNA at altering the properties of calf thymus pol δ. These results therefore suggest that in addition to the interdomain connnector loop, other regions of PCNA are also important effectors of pol δ activity. They also provide a means to couple operationally, the considerable power of in vivo genetic analyses performed in Drosophila with the sophistication of mammalian biochemistry. Results To study the role of the interdomain connector loop of PCNA (amino acids 119-133), we compared human and Drosophila homologs. Of the 15 interdomain connector loop residues, nine are identical between the two; identical residues are shaded (Fig. 1A ). Overall, Drosophila PCNA is >70% identical to that from mammals (e.g., humans; see [ 13 ]). Others showed that PCNA residues 123, 126, 127 and 128 were essential for interaction with pol δ [ 28 ]. Of these four, only one (residue 123) differs between flies and humans. Also shown is a model constructed from the X-ray crystallographically determined structure of PCNA indicating the locations of the sites to be mutated in Drosophila PCNA (Fig. 1B ). Shown (Fig. 1B ) is the X-ray crystal structure of human PCNA. The Drosophila homolog is assumed to be similar. Figure 1 Structure and structural rationale for mutating Drosophila PCNA. A: amino acid sequences of the interdomain loops of Drosophila (designated D.m.) and human (designated H.s.) PCNA. Gray boxes indicate amino acids identical for both organisms; arrows show amino acids thought essential for interaction of human PCNA with human pol δ. Amino acid 123 is the only one which is both essential and different in Drosophila versus human PCNA. B: the "front" side of the human PCNA trimer. Amino acids 119-133 of the interdomain loops are highlighted by showing their α-carbon atoms as black spheres. The α-carbon atom of Val123 is shown as a larger gray sphere. Purification of wild-type and site-specifically mutated PCNA Four NH 2 -terminally his-tagged PCNA variants were highly purified; purity for each is shown (Fig. 2 ). First constructs were prepared encoding 1) NH 2 -terminally his-tagged wild-type human PCNA; 2) NH 2 -terminally his-tagged wild-type Drosophila PCNA (dPCNA) and two dPCNA derivatives; 3) one in which amino acid 123 was mutated from glutamine to valine (Q123V dPCNA); and 4) the other, in which Drosophila amino acids 119-133 were replaced by the corresponding human sequence (dr119-133h dPCNA). Then all four were transformed separately into E. coli (strain M15 [pREP4]) and respective proteins were expressed. Finally bacteria were lysed and his-tagged proteins were purified using various procedures including Ni 2+ -IDA Sepharose chromatography. The purity of each was determined by SDS-PAGE and is shown as indicated (Fig. 2 ). The identity of wild-type human PCNA was confirmed using mouse monoclonal anti-mammalian PCNA antibody PC10; the identity of wild-type Drosophila PCNA was confirmed using affinity purified polyclonal anti- Drosophila PCNA antibodies prepared in rabbits [ 12 ] (not shown). Figure 2 SDS-PAGE analysis of his-tagged PCNA purified from E. coli extracts after regulated bacterial expression. Purification and SDS-PAGE were as described (Experimental Procedures). Lane 1, 0.4 μg wild-type human PCNA was subjected to electrophoresis. Lane 2, 0.8 μg wild-type Drosophila PCNA was subjected to electrophoresis. Lane 3, 0.8 μg Drosophila PCNA containing valine substituted for glutamine at position 123 was subjected to electrophoresis. Lane 4, 0.45 μg Drosophila PCNA containing amino acids 119-133 substituted with the corresponding human PCNA amino acids was subjected to electrophoresis. Migration positions of molecular mass standards are indicated to the right of the figure. Stimulation of calf thymus pol δ activity by highly purified wild-type versus selected mutant PCNA fractions Calf thymus pol δ (apparently homogeneous two-subunit form; see [ 24 ]) was purified and assayed for polymerase activity in the presence of varying concentrations of both highly purified wild-type and specific mutant PCNA molecules. We showed previously that either calf thymus or human PCNA could be used interchangeably as stimulatory co-factors for calf thymus pol δ [ 29 ] (see also [ 12 , 18 , 19 ]). Assays were performed using poly(dA)-oligo(dT) as described (Experimental Procedures). As can be seen, human PCNA resulted in robust stimulation of calf thymus pol δ; much less stimulation was observed for wild-type Drosophila PCNA (Fig. 3 ). Mutation of Drosophila PCNA resulted in substantially increased stimulation of calf thymus pol δ; both substitution of a single amino acid (Q123V dPCNA) and replacement of the entire fly interdomain connector loop with corresponding human amino acids (dr119-133h dPCNA) had demonstrable effects. Of note, at relatively high concentrations, Drosophila PCNA but with the entire fly interdomain connector loop replaced by corresponding human amino acids (dr119-133h dPCNA) was similarly effective to wild-type human PCNA at stimulating the activity of calf thymus pol δ; however, it was considerably less effective at lower concentrations (Fig. 3 ). This suggests an effect on binding of PCNA to pol δ and/or on mutant PCNA multimerization. Figure 3 Effect of various purified PCNA fractions on the DNA polymerase activity of calf thymus pol δ. Calf thymus pol δ was incubated in a reaction mixture as described (see Materials and Methods) for 5 min at room temperature. Each incubation contained 10 ng of pol δ. DNA product synthesized was determined after placing 5-μl aliquots on Whatman DE-81 filters and subsequently washing with a 5% (w/v) solution of Na 2 HPO 4 •12H 2 O. Radioactivity retained on filters was then determined by liquid scintillation counter. Reaction mixtures contained increasing amounts, as indicated on the abscissa, of various PCNA samples, also as indicated. The effects of highly purified wild-type versus selected mutant PCNA fractions on the processivity of incorporation by calf thymus pol δ To examine further, the stimulation of calf thymus pol δ by both wild-type and specific mutant PCNA molecules, we examined effects on processivity of nucleotide incorporation. Processivity is defined as the number of deoxyribonucleotides incorporated each time a DNA polymerase binds its template-primer. As can be seen, without PCNA (Fig. 4 lane 1), pol δ is essentially a distributive enzyme incorporating only a few nucleotides as a result of each binding event. With increasing concentrations of wild-type human PCNA (concentrations increasing from right to left as indicated), processivity of incorporation increases dramatically (Fig. 4 lanes 2–4). This correlates quite closely with the PCNA-mediated activity increase (see Fig. 3 ). Wild-type Drosophila PCNA had relatively much less effect on the processivity of calf thymus pol δ (Fig. 4 lanes 5–7; concentrations again increasing from right to left as indicated). This is also consistent with activity data presented herein (Fig. 3 ) as well as with results reported previously [ 12 ]. When mutants of Drosophila PCNA were tested, both Q123V dPCNA (Fig. 4 lanes lanes 8–10; concentrations again increasing from right to left as indicated) and dr119-133h dPCNA (Fig. 4 lanes lanes 11–13; concentrations again increasing from right to left as indicated), promoted increased pol δ processivities, again consistent with increased activities (Fig. 3 ). Increases were concentration-dependent, also as expected. Figure 4 Effect of various purified PCNA fractions on the processivity of nucleotide incorporation by calf thymus pol δ. Incorporation of [α 32 P]dTMP by calf thymus pol δ was monitored by standard denaturing PAGE. The substrates used were (dA) ~500 -(dT) 12–18 as template-primer and [α- 32 P]dTTP. Concentrations of PCNA, both wild-type and mutant proteins, are as indicated. h, human; dr, Drosophila melanogaster. NH 2 -terminally his-tagged-PCNA fractions are as indicated; wt, wild-type; Q123V, recombinant Drosophila PCNA containing a single amino acid, glutamine at position 123, changed to valine; dr119-133h, recombinant Drosophila PCNA containing the entire interdomain connector loop (amino acids 119-133) replaced with the corresponding human PCNA amino acids. Stable complex formation among pol δ, 32 P-labeled oligonucleotide template-primer and highly purified wild-type versus selected mutant PCNA fractions PAGE band mobility shift assays were used to evaluate, in an essentially qualitative manner, the stability of complex formation among calf thymus pol δ, labeled template-primer and highly purified wild-type versus selected mutant PCNA molecules. As can be seen, wild-type Drosophila PCNA promoted almost no pol δ•PCNA•template-primer complex formation (Fig. 5 ). In contrast, complex-formation with both Drosophila PCNA mutants (Q123V dPCNA and dr119-133h dPCNA) was readily detectable but neither gave results as robust as those seen with wild-type human PCNA (Fig. 5 ). Figure 5 Effect of various purified PCNA fractions on calf thymus pol δ•PCNA• 32 P-labeled oligonucleotide template-primer complex formation. Complex formation among pol δ, various purified PCNA fractions and 32 P-labeled synthetic oligonucleotide template-primers (30-21-mers) was monitored by standard non-denaturing PAGE-band-mobility-shift assays [32]. Each incubation contained 10 ng of pol δ, 70 ng of PCNA and 0.1 pmol/reaction (useable 3'-OH) of annealed template-primer. NH 2 -terminally his-tagged-PCNA fractions are as indicated; wt, wild-type; Q123V, recombinant Drosophila PCNA containing a single amino acid, glutamine at position 123, changed to valine; dr119-133h, recombinant Drosophila PCNA containing the entire interdomain connector loop (amino acids 119-133) replaced with the corresponding human PCNA amino acids. DNA synthesis beyond chemically defined template base lesions promoted by highly purified wild-type versus selected mutant PCNA fractions As a final test, we examined the abilities of various PCNA fractions to promote pol δ-dependent DNA synthesis beyond template base lesions (TLS). PCNA-dependent TLS by pol δ was first reported by O'Day et al. [ 30 ] and subsequently analyzed in detail biochemically [ 29 ]. The structure of the synthetic oligonucleotide used for evaluation is shown in Fig. 6A . For the data shown (Fig. 6B ), X represents the model abasic site (hereafter termed the abasic site [ 31 ]) used previously for many of our studies (e.g., see [ 29 ]). The mobility of the labeled 21-mer primer, PAGE-purified but without any subsequent enzymatic incubation is shown (Fig. 6B lane 1). When calf thymus pol δ alone was added, primer extension opposite the template abasic site was detected but there was no discernible elongation of the resulting 22-mer primer and no full-length product (30-mer) was observed; some degradation of the 21-mer primer, presumably resulting from the activity of the intrinsic pol δ 3'-5' exonuclease, was seen (Fig. 6B lane 2). Addition to incubations of wild-type Drosophila PCNA resulted in slight but readily detectable DNA synthesis beyond the template abasic site; this included some full-length 30-mer (Fig. 6B lane 3). Relatively more full-length 30-mer was seen when Q123V mutant Drosophila PCNA was included in addition to calf thymus pol δ (Fig. 6B lane 4) and still more full-length 30-mer was seen when dr119-133h Drosophila PCNA was added (Fig. 6B lane 5). Clearly, the greatest amount of full-length 30-mer product was seen when wild-type human PCNA was incubated with calf thymus pol δ (Fig. 6B lane 6). Of note, wild-type human PCNA also promotes the tightest complex formation between calf thymus pol δ and 32 P-labeled template-primer DNA (see Fig. 5 ). Figure 6 Effect of various purified PCNA fractions to promote nucleotide incorporation by calf thymus pol δ beyond chemically defined template base lesions. A: the structure of the 5'- 32 P-labeled 30-21-mer template-primer; only the primer (21-mer) was radiolabeled and X indicates the position of a modified tetrahydrofuran moiety (model abasic site) on the 30-mer template. B: lane 1, gel-purified primer alone was subjected to electrophoresis; lanes 2–6, incubations were formulated as indicated with the template-primer shown in A followed by standard denaturing PAGE. h, human; dr, Drosophila melanogaster. For lanes 2–6, each incubation contained 0.5 pmol of labeled primer (3'-OH ends) annealed to 0.5 pmol of template (3'-OH ends), 10 ng pol δ and 70 ng PCNA as indicated. NH 2 -terminally his-tagged-PCNA fractions are as indicated; wt, wild-type; Q123V, recombinant Drosophila PCNA containing a single amino acid, glutamine at position 123, changed to valine; dr119-133h, recombinant Drosophila PCNA containing the entire interdomain connector loop (amino acids 119-133) replaced with the corresponding human PCNA amino acids. Discussion Although human PCNA and Drosophila PCNA are more than 70% identical at the level of primary amino acid sequence, wild-type Drosophila PCNA is only a very poor substitute for human PCNA in cell-free reactions with calf thymus pol δ. This is documented both in this report and previously [ 12 , 32 ]. However, mutating only a single Drosophila PCNA amino acid, glutamine at position 123 (Q123) to valine (V), leads to a dramatic enhancement in the abilities of Drosophila PCNA to stimulate calf thymus pol δ. Effects were shown on total activity (Fig. 3 ), processivity (Fig. 4 ), pol δ•PCNA•template-primer complex formation (Fig. 5 ) and extended DNA synthesis beyond a template abasic site (Fig. 6 ). Replacing the entire interdomain connector loop of Drosophila PCNA (amino acids 119-133) with the corresponding residues from human PCNA resulted in additional enhancement (Figs. 3 , 4 , 5 , 6 ), but in neither case were the mutants of Drosophila PCNA (Q123V dPCNA or dr119-133h dPCNA) equivalent to wild-type human PCNA in the stimulation of calf thymus pol δ. Our data indicate that although a single Drosophila PCNA amino acid at position 123 (in addition to conserved residues 126–128) is very important for pol δ-stimulation, the further enhancement of stimulation seen when the entire interdomain connector loop of Drosophila PCNA (amino acids 119-133) was replaced with the corresponding residues from human PCNA suggests that other residues in this loop are also involved directly in binding pol δ. Alternatively, it is possible that loop residues other than 123 and 126–128 play a secondary or indirect (e.g., conformational) role in positioning crucial amino acids so as to optimize their direct binding to pol δ. In this context, we would like to call attention to the fact that at relatively low concentrations, dr119-133h dPCNA is considerably less effective than wild-type human PCNA in stimulating the activity of calf thymus pol δ; at higher concentrations, dr119-133h dPCNA and wild-type human PCNA stimulate calf thymus pol δ similarly. This implies complex protein-protein interactions between PCNA and pol δ such that biochemical properties recorded in dilute solutions in vitro may not accurately predict properties manifest at much different and generally much higher intranuclear concentrations present in vivo . Alternatively, PCNA must be present as a trimer (three-subunit ring) in order to function. Since the equilibrium among monomer, dimer and trimer was shown to depend on PCNA protein concentration [ 33 ], it is certainly possible that the difference observed between dr119-133h dPCNA and wild-type human PCNA actually reflects differences in the K eq for PCNA multimerization. These two possibilities, concerning both complicated pol δ•PCNA interactions and PCNA multimerization, are not mutually exclusive. Similarly, the fact that replacement of the entire interdomain connector loop of Drosophila PCNA (amino acids 119-133) with the corresponding residues from human PCNA did not result in a molecule as effective in stimulating calf thymus pol δ as human PCNA suggests that regions other than the interdomain connector loop are important for pol δ-stimulation. Our data do not address the question of whether these putative "other regions" affect pol δ directly (e.g., like the interdomain loop) or indirectly (e.g., through conformational effects on other regions of the molecule that do bind pol δ directly). Additional mutagenesis studies may shed light on this issue. For example, based on experiments of others, it seems likely that the extreme C-terminus of PCNA also interacts directly with pol δ (see [ 23 , 34 - 36 ]). Hence it may be of interest to perform similar mutagenesis experiments to those reported here, focusing instead on the C-terminal region of Drosophila PCNA, rather than the interdomain connector loop. We think it should also be noted that both Oku et al. [ 35 ] and Ola et al. [ 36 ] prepared hybrid proteins between human and S. cerevisiae PCNA. As in our studies, Ola et al. [ 36 ] found that regions other than the interdomain connector loop of PCNA were important for interaction with pol δ. These authors suggested that additional interacting regions were likely to exist both in the PCNA C-terminus and N-terminus. It may also be of interest to prepare double-mutants, first in the interdomain connector loop of Drosophila PCNA, thereby allowing efficient in vitro function with purified calf thymus pol δ, and then elsewhere in the PCNA molecule corresponding to interesting sites defined phenotypically by in vivo genetic studies of others. For example, it might be possible to determine if particular mus 209 mutations leading to enhanced mutagen sensitivity among affected organisms (see [ 37 ] and references therein) alter any functional interactions between PCNA and pol δ in vitro . Results of such studies could lead to novel biochemical insights regarding the mechanism(s) by which point mutations in the Drosophila PCNA gene lead to enhanced mutagen sensitivity among animals bearing these mutations. The strategy taken here will presumably allow study of interactions between PCNA and other proteins with which it interacts. In this context, we think it important to note that partial effects on pol δ-stimulation have been recorded. This suggests that our methodology will also allow detection of partial rather than complete effects on the binding of other proteins. Interactions between PCNA and many of the molecules with which it interacts have recently been mapped [ 23 ] and for example, one might immediately compare interactions between several mammalian proteins (e.g., human RF-C, DNA ligase I, FEN I and/or p21) and both various wild-type and mutant PCNA molecules described in this paper. Functional (e.g., effects on pol δ activity) as well as direct binding measurements may be made. As with PCNA•pol δ interactions, it may ultimately be feasible to correlate interesting PCNA molecules defined phenotypically using genetic analyses performed in living animals and biochemical studies of specific PCNA•protein binding. For example, do mutagen sensitive mus 209 animals bear mutations in a region of PCNA responsible for MSH binding? Both MSH3 and MSH6 were reported to possess a consensus motif for binding to the interdomain connector loop of PCNA [ 38 ]. Finally, we think it important to note that pol δ has most recently been reported to contain at least four subunits (see e.g., [ 39 , 40 ]) yet all experiments performed here were with the two-subunit form of the enzyme purified from calf thymus. We and others have shown that the larger subunit, p125, is catalytic while the smaller, p50, does not seem to contact the DNA closely (see e.g, [ 41 ]), but instead, is required for processivity-stimulation by PCNA (e.g., see [ 42 ]) to which it apparently binds. It is also clear that PCNA binds to what has been termed, the third pol δ subunit, p68 or p66 in mammalian systems [ 39 , 43 , 44 ], Cdc27p in S. pombe [ 40 ] and Pol32p in S. cerevisiae [ 45 , 46 ]. Clearly the physiologically important interaction between PCNA (either mutant or wild-type) and this third pol δ subunit was omitted from our analyses, but could markedly affect any or all of the responses of polymerase to PCNA that we reported here. Conclusions Through our experiments, we showed that Drosophila PCNA could be "humanized" and that "humanization" (mutation of key Drosophila residues to human ones) increased effects on mammalian pol δ. The highly purified two-subunit form of pol δ was used for all of our studies. It is possible, though we think it unlikely, that different conclusions would be reached if a different form of pol δ (three-or four-subunit) was used. Nevertheless two of the effects we observed could be considered beneficial. They were enhancement of polymerase activity and processivity. A third effect seems likely to be detrimental, at least over the long term, that is increased synthesis opposite and beyond a chemically defined template base lesion (TLS). Our data suggest that all three of these effects result from enhancement of PCNA-dependent stability of the pol δ•PCNA•template-primer complex. In other words, in the range that we have studied, the more tightly pol δ binds to DNA, the greater its activity, the greater its processivity, but also the more likely it is to catalyze TLS. Our results provide an explicit approach to correlate in vivo genetic studies with rigorous in vitro biochemistry. Methods Materials Unlabeled deoxyribonucleoside triphosphates (dNTPs) were from Boehringer-Mannheim; [α- 32 P]ATP and [α- 32 P]dTTP were from Amersham Corp. E. coli DNA polymerase I Klenow fragment without 3'-5' exonuclease activity (exo-), was expressed and purified according to standard protocols [ 47 ]. Terminal deoxynucleotidyl transferase (TdT) was from Sigma. Micrococcal nuclease was from Boehringer-Mannheim. Pfu DNA polymerase was from Stratagene. Ni 2+ -IDA Sepharose was from Pharmacia (Piscataway, NJ). Acrylamide and methylene bis-acrylamide were from Eastman Organic Chemicals and for protein SDS-PAGE, were further purified by adsorption of impurities to activated charcoal. For PAGE of nucleic acids, they were purified by adsorption to an ion exchange resin. All other materials were of reagent grade and were used without additional purification. Proteins PCNA was purified to apparent homogeneity from calf thymus [ 17 ] as was pol δ [ 24 , 48 ]. Human PCNA cDNA was cloned into a bacterial expression vector and human PCNA was purified from an E. coli extract, also to apparent homogeneity [ 18 ]. D. melanogaster PCNA was purified to apparent homogeneity identically after bacterial expression [ 13 ]. A his-tag was added to the NH 2 -termini of both human and Drosophila PCNA by cDNA insertion into pQE30 (Qiagen, Valencia, CA) using Bam H1 and Hind III restriction endonuclease sites. Nucleic acids Templates and primers, all of defined sequence, were synthesized conventionally by Dr. F. Johnson and colleagues (Stony Brook). Before use, they were purified by standard denaturing PAGE [ 49 ]. All other DNA manipulations were performed according to standard techniques [ 49 ]. Methods Much of the methodology was described in detail previously [ 12 , 19 , 20 , 24 , 29 , 32 , 41 , 50 , 51 ]. SDS-PAGE was according to Laemmli [ 52 ] as modified [ 53 ] on minigels or as reported previously [ 54 ]. For immunoblots, proteins were transferred electrophoretically to nitrocellulose [ 55 ] and resulting replicas were probed with antibodies. Reactivity was visualized colorimetrically [ 56 ] with alkaline phosphatase-conjugated goat anti-IgG antibodies [ 57 , 58 ] and a one-solution phosphatase substrate (Kirkegaard and Perry, Gaithersburg, MD). Immunologic detection of human PCNA was with mouse monoclonal antibody (mAb) PC10 (Oncogene Sciences, Uniondale, NY). Detection of Drosophila PCNA was with affinity purified polyclonal rabbit anti- Drosophila PCNA antibodies [ 12 ]. Restriction endonucleases were from Boehringer (Indianapolis, IN) and were used according to the vendor's instructions. DNA sequencing performed in both directions was according to Sanger et al. [ 59 ] using a fluorescence-based method and an ABI 373 (Applied Biosystems, Foster City, CA) automated DNA sequencer. Site-directed mutagenesis of Drosophila PCNA Site-directed mutagenesis of NH 2 -terminally his-tagged Drosophila PCNA was performed exactly as described [ 20 ] to generate either the Q123V protein or chimeric molecules containing the entire Drosophila PCNA sequence except for amino acids 119-133 which were replaced by the corresponding residues from human PCNA. Purification of his-tagged PCNA Purification of his-tagged PCNA to apparent homogeneity was performed exactly as previously described [ 20 ]. Characterization was by SDS-PAGE (Fig. 2 ) and immunoblot analysis. DNA polymerase δ incubations Assays of pol δ on synthetic oligonucleotide template-primers were performed essentially as previously described [ 24 ]. Primers were 5' end-labeled with T4 polynucleotide kinase in the presence of [γ- 32 P]ATP. Afterward, labeled primer was annealed to an unlabeled template. The standard reaction mixture for pol δ contained 40 mM Bis-Tris, pH 6.7, 6 mM MgCl 2 , 1 mM dithiothreitol, 10% glycerol and 40 μg/ml bovine serum albumin. Additional details are provided in the figure legends. Incubations were terminated by addition of standard stop solution and aliquots were subjected to 12% PAGE in the presence of 7 M urea and 15% formamide. After electrophoresis, gels were subjected to autoradiography and/or Molecular Dynamics 445 SI PhosphorImager analyses. Pol δ processivity Processivity was evaluated qualitatively using (dA) ~500 annealed to (dT) 12–18 (both from Pharmacia) in a final volume of 5 μl containing 6 nmol poly(dA) (nucleotide), 0.2 nmol (dT) 12–18 (nucleotide), 10 μM dTTP, 100 μCi [α- 32 P]dTTP, 40 mM Bis-Tris, pH 6.7, 6 mM MgCl, 1 mM dithiothreitol, 10% glycerol, 40 μg/ml bovine serum albumin, 10 ng of highly purified pol δ and various quantities of different PCNA samples as indicated. Assays were for 5 min at room temperature and were stopped by addition of standard PAGE stop solution and PAGE in the presence of 7 M urea. After electrophoresis, gels were subjected to autoradiography and/or Molecular Dynamics 445 SI PhosphorImager analyses. Nondenaturing PAGE band mobility shift assays Nondenaturing PAGE band mobility shift assays were performed essentially as previously described [ 32 ] but without MgCl 2 and otherwise as detailed in the figure legend. EDTA was included in each incubation and in the gel electrophoresis buffer at a final concentration of 3 mM. Authors' contributions DJuM performed all enzymologic and mobility shift assays with DNA polymerase δ in combination with both wild-type and various mutant PCNA molecules. He also designed, engineered and characterized all recombinant PCNA molecules. DJuM expressed several recombinant proteins in bacteria and purified them. Finally, he participated in DNA polymerase purification and drafted the original manuscript. MM expressed some recombinant proteins in bacteria and purified them. She also purified and characterized most DNA polymerase substrates. HM participated in DNA polymerase purification and manuscript preparation. PAF advised DJuM on execution and interpretation of experiments and assisted both in figure design and all other aspects of manuscript preparation. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC515284.xml |
514603 | Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors | Background RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death. Results As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing. Conclusions Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems. | Background Gene silencing is a powerful tool with which to study protein function. Gene inactivations in mice have revolutionised the way we study both basic biology and a plethora of disease types [ 1 , 2 ]. Gene silencing in human cells has, until recently, proven difficult to achieve [ 3 ]. Research with plants, flies and worms recently uncovered a mechanism by which eukaryotic cells target mRNAs, and perhaps even genetic loci, for specific gene silencing. This process is termed RNA interference (RNAi). RNAi can also be induced in mammalian cells using double-stranded RNAs (dsRNAs), and has become the method of choice for targeted knock-down of gene expression in mammalian cells [ 4 ]. The apparent specificity of RNAi also enables allele-specific gene targeting [ 5 ]. Initial studies using RNAi in mammalian cells centred around transient knock-down of target gene expression, either using direct transfection of synthetic short interfering RNA (siRNA) [ 6 ], transfection of in vitro transcribed siRNA [ 7 ] or transient expression of short dsRNA via transfection of plasmid DNA bearing RNA Polymerase III promoter-driven expression cassettes [ 8 , 9 ]. Short dsRNAs of 19 to 29 base-paired nucleotides, complementary to the target mRNA, were expressed as 2 complementary RNAs or as a hairpin structure (shRNA), and resulted in knock-down of the target message. While these initial RNAi methods gave a rapid phenotypic read-out in vitro, stable knock-down of gene expression is required for monitoring long-term effects on cell function, for example, in developing tumors in vivo or in cell-based gene therapy approaches. Efficient delivery of RNAi-inducing dsRNA or expression cassettes is required for effective transient and long-term studies. Transfer of functional shRNAs using lentiviral vectors appears to be a valid approach for effective, stable construct delivery to both cell lines [ 10 ] and primary cells [ 11 - 13 ]. More recently, using several different expression systems and target cells, reports have highlighted the utility and specificity of the RNAi approach [ 14 - 17 ]. Maintaining RNAi-inducing dsRNA below 30 nucleotides in length was thought to avoid activation of the interferon-induced anti-viral response. PKR is a key anti-viral regulator and its expression can be induced by the interferon response [ 18 ]. PKR is activated when bound to dsRNA longer than 30 nucleotides, despite interacting with shorter dsRNA molecules [ 19 ]. Four recent reports have pointed towards limitations to using RNAi as a tool in mammalian cells. The first demonstrated off-target gene silencing [ 20 ], highlighting the redundancy of short nucleotide sequences in the human transcriptome. The second characterised the expression profile of genes as a result of lentiviral vector-mediated RNAi. Interferon regulated gene expression was increased even with dsRNAs as short as 19 nucleotides [ 21 ]. The third report demonstrated similar interferon response gene up-regulation, after transfection of cell lines with synthetic siRNAs as short as 21 nucleotides [ 22 ]. Finally, Scacheri et al documented significant siRNA sequence-dependent changes in the expression of non-targeted proteins [ 23 ]. In this work we used a simple approach for gene silencing in human tumor cell lines, using lentiviral vectors for stable delivery of shRNAs. We aimed to study the effects of targeting the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA on cell survival in the presence of pro-apoptotic stimuli. In addition to its inhibitory activity on the urokinase plasminogen activator, PAI-2 is thought to have anti-apoptotic properties [ 24 , 25 ]. Its molecular targets in this respect are unknown. A recent report demonstrated a functional interaction between PAI-2 and the retinoblastoma protein cell cycle regulator [ 26 ]. Using lentiviral vectors for delivery of RNAi-inducing expression cassettes we achieved potent PAI-2 gene silencing, accompanied by a rapid cytotoxic effect. The degree of cytotoxicity was proportional to shRNA length and induction of an interferon response gene could be detected when shRNA of 21 complementary base pairs or more was expressed. The phenotype was not target gene specific, as PAI-2 overexpression failed to rescue cytotoxicity and control hairpins were also cytotoxic. Using lower vector titre, reduced shRNA expression and interferon response induction was measured without compromising gene silencing. Using a 19 complementary base pair shRNA expression vector, which reduced PAI-2 expression and induced no initial cytotoxicity or interferon response, transduced cell marker gene expression was maintained but gene silencing lost in long-term cell culture. Our results highlight the need for careful controls to monitor specificity and maintenance of gene silencing when using RNAi for stable loss-of-function studies in mammalian cells. Results Efficient transfer of RNAi-inducing cassettes using lentiviral vectors Lentiviral vectors were generated which deliver an expression cassette for human U6 promoter-driven expression of short hairpin RNA (shRNA), with exact homology to the human PAI-2 mRNA. The vector expression cassette also bears the enhanced green fluorescent protein (GFP) gene under the control of the EF-1α promoter, and an internal ribosome entry site (IRES) sequence (see Figure 1A ). This cassette allows permanent expression of GFP in transduced cells, and the possibility of concomitant overexpression of a further cDNA, between the EF-1α promoter and IRES sequences, not used here. The shRNA sequences were chosen from the PAI-2 mRNA to include a 5' guanosine at the U6 promoter transcriptional start site, to exclude the 5' and 3' 100 nucleotides of the PAI-2 open reading frame, and to be between 30 and 70 % guanosine/cytidine rich. As controls, we have used a vector leading to expression of GFP alone (EGFP), a vector with the U6 promoter and transcriptional termination signals but lacking a hairpin encoding sequence (U6PT), and vectors leading to expression of scrambled sequences of certain hairpins. Figure 1 demonstrates the efficient transduction of Isreco-1 (IS-1) human colorectal carcinoma cells with one such PAI-2 targeting vector (sh325). Sh325 is designed for expression of a shRNA with a 25 nucleotide double-stranded stretch (a 25 mer) to target the PAI-2 mRNA. As controls, we used U6PT and a scrambled sequence (sh325scr) vector. Four days after transduction, each cell population expressed high levels of GFP, as a marker for transduction (Figure 1B ). Compared to non-transduced cells, or cells transduced with the U6PT control vector, we measured a clear knock-down of endogenous PAI-2 protein and mRNA in cells transduced with the sh325 vector (see figure 1C and 1D ). Figure 1 Effective gene silencing using lentiviral vectors for RNAi. The gene transfer cassette common to each vector for RNAi is shown in A. Each construction for RNAi was designed for expression of a shRNA, homologous to the target mRNA or with a scrambled sequence, driven by the RNA polymerase III-controlled human U6 promoter and ending with a terminator (T) sequence. The shRNA is represented by two arrows which encode 19 to 25 nucleotide complementary sequences and are joined by an eight nucleotide loop (L). EGFP expression is via the EF-1α promoter, oriented in the opposite direction, driving an IRES sequence and the EGFP gene. Each cassette is flanked by the HIV long terminal repeats (LTR), of which the 3' LTR is modified to ensure that the vectors are self-inactivating upon integration (SIN). B shows flow cytometry analysis of non-transduced IS-1 cells and cells four days after transduction with the U6PT control vector, a vector for expression of shRNA complementary to a region of the PAI-2 mRNA (sh325) and a vector for expression of a shRNA with a scrambled sh325 sequence (sh325scr). C shows an immunoblot for detection of PAI-2 in the cell lysate of these cells. NS highlights a single non-specific band which is consistently detected in PAI-2 immunoblots using IS-1 cell lysates. In D, PAI-2 mRNA levels from the same samples are measured by QRT-PCR of cDNA, using the ΔCT method and hypoxanthine phosphoribosyl transferase (HPRT) as the control gene. Each target gene was detected in duplicate, error bars represent the standard deviation of mean values. However, the control vector with a scrambled sequence (sh325scr) also reduced PAI-2 mRNA and protein levels. Equal sample loading for immunoblots was confirmed by Ponceau S staining of nitrocellulose membranes (data not shown) and the presence of equal amounts of a PAI-2 monoclonal antibody-reactive non-specific band in each blot (NS in Figure 1C ). Rapid cytotoxic effect of RNAi vectors Many of the initial loss-of-function studies using RNAi have measured the phenotypic effect of gene silencing in the immediate time frame after introduction of the siRNA or RNAi-inducing expression vector. As seen in Figure 1 , four days after transduction with our vectors appears to be sufficient for efficient target gene silencing. Transduction with lentiviral vectors leads to stable long-term integration of the desired transgene cassette, a key advantage in their use compared to other transient or less stable expression systems. Thus we reasoned that transduction with RNAi-inducing cassettes, using lentiviral vectors, would also be stable unless the reduction in target gene expression gave transduced cells a significant growth disadvantage or cytotoxic phenotype. Four to five days after transduction, IS-1 cells bearing the sh325 construct or cells transduced with a scrambled sh325 sequence rapidly changed morphology, compared to U6PT-transduced control cells. Sh325-transduced cells began to disintegrate into small particles and detach from cell culture dishes. After 10 days most of the sh325-transduced cells were dead while the U6PT-transduced cells were growing like the parent cell line. The scrambled hairpin vector-transduced cells gave a weaker cytotoxic phenotype, with deteriorating cell morphology and some detachment of transduced cells. Figure 2A shows the morphology of IS-1 cells 6 days after transduction. To understand further this cytotoxic effect, we performed quantitative RT-PCR (QRT-PCR) on RNA isolated from IS-1 cells, 4 days after transduction with the same vectors, in order to measure the levels of the 2'5'-oligoadenylate synthetase-1 (OAS1) mRNA after transduction with each vector. The OAS1 gene is recognised as an interferon response gene and has also been monitored elsewhere when using RNAi [ 21 ]. We measured increases in OAS1 expression in both sh325 and scrambled sh325 vector-transduced cells, whereas control-transduced cells (U6PT) had equal OAS1 levels to non-transduced cells (see Figure 2B ). Figure 2 Cytotoxicity and OAS1 induction with RNAi vectors. In A, morphology was observed using phase contrast microscopy of non-transduced IS-1 cells, or cells six days after transduction with vectors leading to expression of no shRNA (U6PT), a 25 mer shRNA targeting PAI-2 (sh325) and a scrambled 25 mer control shRNA (sh325scr). B shows comparison of OAS1 expression in non-transduced cells or cells four days after transduction with U6PT, sh325 and sh325scr vectors, by QRT-PCR. Each target gene was detected in duplicate, error bars represent the standard deviation of mean values. 19 mer shRNAs induce less cytotoxicity than longer hairpins and do not increase OAS1 expression As both the target gene-specific and the scrambled sequence 25 mer shRNAs, sh325 and sh325scr, induced the OAS1 interferon response gene, we generated further vectors for delivery of shRNAs with reduced hairpin length. We reduced the length of sh325, from 25 to 23, 21 and 19 nucleotides and named the novel vectors sh323, sh321 and sh319, respectively. The truncations were made at the 3' end of the 25 nucleotide sense strand and therefore the 5' of its complementary anti-sense sequence (see Table 1 ). Each vector was used to transduce IS-1 cells and the growth of GFP positive cells monitored at 4 and 10 days after transduction, compared to U6PT control-transduced cells (see Figure 3A ). Targeting of the PAI-2 mRNA and protein was monitored, four days after transduction, by QRT-PCR and immunoblotting of cell lysates (Figure 3B and 3C ). While each PAI-2 targeted vector successfully reduced PAI-2 mRNA and protein four days after transduction, a strong negative selection was seen for shRNA-expressing cells after a further six days of culture. This selective pressure on transduced cells was stronger with the 21 mer, 23 mer and 25 mer shRNAs than with the shorter sh319-derived 19 mer (Figure 3A ). OAS1 mRNA levels were measured by QRT-PCR of transduced cell cDNA four days after transduction (Figure 3B ). The cells transduced with the 21 mer, 23 mer and 25 mer shRNAs showed induction of OAS1 mRNA, however, contrary to our expectations, highest OAS1 levels were obtained with the 21 mer shRNA. Loss of GFP positive cells over time was comparable for 21 mer, 23 mer and 25 mer hairpin constructs. To determine whether the lack of OAS1 induction was specific to sh319 or common to other 19 mer shRNAs, further transductions and QRT-PCR analysis were performed on mRNA from IS-1 cells transduced with sh319, sh321 and sh319scr vectors. sh319scr encodes a shRNA with a scrambled sh319 sequence. This analysis also confirms the specificity of the PAI-2 silencing, by comparing sh319 to sh319scr. Figure 3D shows that no induction of OAS1 was measured using sh319 or sh319scr vectors and sh319scr had no effect on the PAI-2 mRNA level. Table 1 Construct details and shRNA sequences. Vector names and the shRNA sequences they encode. In comments, numbers are coding human PAI-2 mRNA nucleotides (adapted from accession number M18082). Hairpin sequence Name sense loop antisense Comments sh319 GCGCACACCUGUACAGAUG CAAGCUUC CAUCUGUACAGGUGUGCGC PAI-2 684–702 sh321 GCGCACACCUGUACAGAUGAU CAAGCUUC AUCAUCUGUACAGGUGUGCGC PAI-2 684–704 sh323 GCGCACACCUGUACAGAUGAUGU CAAGCUUC ACAUCAUCUGUACAGGUGUGCGC PAI-2 684–706 sh325 GCGCACACCUGUACAGAUGAUGUAC CAAGCUUC GUACAUCAUCUGUACAGGUGUGCGC PAI-2 684–708 sh319scr GUCAUACCGGCAAGGAUCC CAAGCUUC GGAUCCUUGCCGGUAUGAC scrambled sh319 sh325scr GGCCGGAGAUAAGUUCACUCAACUC CAAGCUUC GAGUUGAGUGAACUUAUCUCCGGCC scrambled sh325 sh119 GAAGACCAGAUGGCCAAGG CAAGCUUC CCUUGGCCAUCUGGUCUUC PAI-2 151–169 EGFP No hairpin, empty vector. none U6PT Human U6 promoter/terminator, no hairpin. none Figure 3 Shorter shRNA length reduced, but did not eliminate, cytotoxicity. A represents flow cytometry analysis of IS-1 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells is shown in each histogram). B shows a comparative analysis of PAI-2 and OAS1 mRNA, in the samples described in A, 4 days after transduction. Data were generated by QRT-PCR and error bars are as described in previous figures. In C, cell lysates from samples of transduced cells described in A and B were subjected to immunoblotting with anti-PAI-2 monoclonal antibodies. Ponceau S staining served as a gel loading control, as did comparison of a single non-specific band (NS) in the immunoblot. D shows QRT-PCR analysis, as in B, for IS-1 cell mRNAs after transduction with or without U6PT, sh319, sh319scr and sh321 vectors. Cytotoxicity is not target gene specific To determine if all or part of the cytotoxic effect seen with our shRNAs was due to down-regulation of PAI-2, we generated an IS-1 cell line which overexpresses functional PAI-2. A lentiviral vector was produced which delivers the wild type PAI-2 cDNA, and used to transduce IS-1 cells. This resulted in a homogeneous population of cells which overexpress PAI-2 (see Figure 4A , IS-1 PAI-2 cells). Using immunoblotting of PAI-2/u-PA complexes, formed by mixing IS-1 PAI-2 cell lysates with low molecular weight u-PA, we demonstrated that this overexpressed protein was functional (see Figure 4B ). We transduced these cells with the series of shRNA-delivering vectors described in Figure 3 (sh325, sh323, sh321 and sh319), to test whether functional PAI-2 overexpression could reverse the cytotoxic phenotype. As even endogenous PAI-2 is not completely silenced using these vectors we reasoned that the RNAi they deliver would not be capable of functionally silencing overexpressed PAI-2. As predicted, our PAI-2 targeting shRNAs were unable to completely reduce the overexpressed PAI-2 protein levels (see Figure 4C , compared to the relative non-specific band intensity in Figure 3C ). However, the cytotoxic effect seen with the parent IS-1 cell line was also clearly apparent in the PAI-2 overexpressing cells. We monitored the loss of GFP positive cells in the transduced PAI-2 overexpressing cell populations and saw almost identical kinetics, compared to the parent cell line (compare Figure 3A and Figure 4D ). These data show that the cytotoxic effect is not target gene specific. We also measured the level of PAI-2 mRNA in this experiment, by QRT-PCR. Despite a several-fold decrease in overexpressed PAI-2 protein level (see immunoblot in Figure 4C ) we were unable to detect knock-down of the overexpressed mRNA (Figure 4E ). In a similar manner to the IS-1 parent cell line, transduction of PAI-2 overexpressing cells with sh325, sh323, sh321, but not the sh319 vector, induced the OAS1 interferon response gene (Figure 4F ). To exclude IS-1 cell-specific effects of our vectors we transduced IS-1 cells and HeLa cells with a GFP control, the sh319 and the sh319 scrambled sequence (sh319scr) vectors. We monitored the percentage of GFP positive cells at day 4, 8 and 11 after transduction and observed similar selective loss of GFP positive cells for the sh319 and sh319scr vectors, in both cell lines (Table 2 ). Figure 4 PAI-2-targeted RNAi with overexpression of functional PAI-2 in IS-1 cells. IS-1 cells were transduced with a lentiviral vector for delivery of the human PAI-2 cDNA under control of the CMV promoter. A shows a flow cytometry analysis for detection of PAI-2 in transduced IS-1 PAI-2, and non-transduced IS-1 cells. Both cell types were fixed, permeabilised and labelled with anti-PAI-2 monoclonal antibodies, then incubated with PE-labelled secondary antibodies. In B, the functional activity of overexpressed PAI-2 was assessed by immunoblotting of cell lysates, for PAI-2 expression, with or without the addition of 10U of u-PA. u-PA alone is included in lane 1. In C, PAI-2 protein levels were assessed in cell lysates from IS-1 PAI-2 cells, after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. NS in B and C highlights a single non-specific band which is consistently detected in PAI-2 immunoblots of IS-1 cell lysates. D represents flow cytometry analysis of IS-1 PAI-2 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells are given in each histogram). E and F show comparative analyses of PAI-2 and OAS1 mRNA expression, respectively, in samples described in D. Samples were analysed 4 days after transduction. Data were generated by QRT-PCR, each target gene was detected in duplicate, error bars represent the standard deviation of mean values. Table 2 Percentage GFP positive cells over time in shRNA-expressing IS-1 and HeLa cells. Transduced cells were assessed for GFP expression by flow cytometry. GFP positive cells were gated equally for each cell type, 4, 8 and 11 days after transduction with EGFP, sh319 and sh319scr vectors. % GFP positive cells IS-1 HeLa Vector Day 4 Day 8 Day 11 Day 4 Day 8 Day 11 EGFP 95 95 93 92 94 93 sh319 86 70 56 79 68 50 sh319scr 90 76 59 82 64 45 Reducing lentiviral vector titre can reduce shRNA expression level and OAS1 induction, while maintaining gene silencing To understand whether lentiviral vector titre and resulting shRNA expression levels influence the non-specific effects described, IS-1 cells were transduced with the U6PT, sh319 and sh321 vectors as described above and also using a 10-fold reduction in vector titre (vector titre details are given in the figure 5 legend). Both sh319 and sh321 vectors effectively down-regulate PAI-2, but only the sh321 vector induces OAS1 expression (see Figure 3B ). Using 10-fold lower vector titre had little impact on PAI-2 mRNA silencing, as seen using QRT-PCR (Figure 5A ), and resulted in a small decrease in OAS1 mRNA induction (Figure 5B ) in sh321-transduced cells. To assess whether lower vector titre resulted in lower shRNA expression, total RNA from cells transduced with different titres of sh321 vector was subjected to RNase digestion after hybridization with a 32 -P labelled RNA probe, designed to protect the first 19 nucleotides of the sh325 shRNA. Upon hybridization, this "sh3" probe should protect short RNA expressed in sh321 vector-transduced cells from RNase digestion. As negative controls, RNA from U6PT-transduced cells was subjected to the RNase protection procedure, and the sh3 probe was treated with RNase without target RNA. As a positive control a probe for the Mir-16 miRNA was constructed and used to detect endogenous Mir-16 miRNA in U6PT-transduced cells using the same protocol (Figure 5C ). Sh3 probe-protected RNA was detected in sh321-transduced cells. Reducing the viral titre clearly reduced the expression of shRNA and this correlated with reduced OAS1 induction. Sh3 probe-protected RNA was not detected in control-transduced cells and the sh3 probe was completely digested in the absence of target RNA (Figure 5C ). This data demonstrates that vector-derived shRNA expression can be reduced without impacting gene silencing and that lower expression correlates with a reduced interferon response. Figure 5 Efficient target gene silencing with reduced OAS1 induction and lower shRNA expression. Total RNA was isolated from IS-1 cells (CTRL) or IS-1 cells 4 days after transduction with U6PT, sh319 or sh321 vectors. After reverse transcription, cDNA was analysed for expression of PAI-2 (A) and OAS1 (B). Data were generated by QRT-PCR, each target gene was detected in triplicate, error bars represent the standard deviation of mean values. 1 ml or 0.1 ml of each lentiviral vector stock was used, hence the designations 1 and 0.1. Vector titres were approximately 10 6 transducing units per ml resulting in a multiplicity of transduction of approximately 10 for 1 ml used or 1 for 0.1 ml used. In C, shRNA expression was detected in total cell RNAs using a modified RNase protection protocol. Total RNA was mixed with radiolabelled probes for hybridization and RNase protection. Samples were resolved on a 15 % Acrylamide/8M Urea/TBE gel and RNase protected probes detected by autoradiography. Lane 1 shows the Mir-16 probe without RNase digestion or target RNA, lane 2 is as lane 1 with RNase digestion and lane 3 as lane 2 with U6PT-transduced cell RNA as hybridization target. Lane 4 shows the sh3 probe without RNase digestion or target RNA, lane 5 as lane 4 with RNase digestion and lane 6 as lane 5 with U6PT-transduced cell RNA as hybridization target. In lane 7 sh321-transduced cell RNA was used as the hybridization target for the sh3 probe with RNase digestion, and lane 8 is as lane 7 except that cells were transduced with 10-fold less vector titre (sh321 1 and sh321 0.1). Known nucleotide lengths (Ntds.) for the probes and the protected Mir-16 endogenous RNA are marked. Loss of long-term gene silencing despite persistent transduction marker gene expression In an attempt to generate a cell line with stable PAI-2 mRNA silencing without interferon response induction, we generated additional vectors which deliver 19 to 25 nucleotide shRNAs targeting different regions of the PAI-2 mRNA. Of these, the sh119 construct reduced PAI-2 expression, did not induce OAS1 and had no effect on cell morphology one week after transduction (data not shown). In parallel, we transduced IS-1 cells with the sh119 vector or a GFP control vector. We achieved high percentage transduction rates which were monitored for over two weeks (Figure 6A ). GFP positive cells, from control- and sh119-transduced populations were sorted twice, using flow cytometry, to further enrich the GFP positive population of each (EGFPs and sh119s). Sh119-transduced cells showed PAI-2 gene silencing 10 days after transduction but not after one month of cell culture (Figure 6B and 6C ). GFP marker gene expression was maintained in both sorted cell populations. During the prolonged culture period we noticed that, while the percentage of GFP positive cells remained stable, the intensity of GFP detected was reduced in the sh119-transduced cells, compared to GFP alone controls (Figure 6A ). This was accompanied by a significant reduction in integrated vector copies (data not shown). Thus, although the marker protein was maintained at a reduced expression level, the prolonged culture period selected against cells with effective gene silencing. This suggests the presence of subtle cytotoxic effects of short dsRNA expression which are not apparent in the initial post transduction period and are in the absence of interferon response gene induction. Overexpression of PAI-2 in the IS-1 cells did not reduce the long-term selective effect on transduced cells or GFP expression levels in sh119-transduced cells (see Table 3 ). Figure 6 Long-term gene silencing is not stable, despite persistent marker gene expression. In A, IS-1 cells transduced with EGFP control or sh119 vectors were analysed by flow cytometry 3 and 17 days after transduction, and compared to non-transduced cells. EGFP expressing cells, from both transduced cell populations, were selected by cell sorting and named EGFPs and sh119s. 39 days after transduction, these cells were analysed for EGFP expression. Percentage EGFP positive cells, assessed by the M1 gating shown, are given in each histogram. In B, PAI-2 expression in EGFP and sh119 vector-transduced cell lysates were analysed by immunoblotting, 10 days after transduction. C is the same PAI-2 immunoblot as B, performed using EGFPs and sh119s cell lysates, 33 days after transduction. Table 3 Percentage GFP positive cells and mean GFP fluorescence in sh119-transduced IS-1 and IS-1 PAI-2 cells. Transduced cells were assessed for GFP expression by flow cytometry. GFP positive cells were gated equally for each cell type, 5 and 31 days after transduction with the sh119 vector. Mean F is the mean fluorescence of gated GFP positive cells. % GFP positive cells Mean F of GFP positive cells Day5 Day31 Day5 Day31 IS-1 cells 85 40 146 83 IS-1 PAI-2 cells 82 46 180 96 Discussion Here we report the use of lentiviral vectors for the delivery of expression cassettes designed for RNAi-induced stable knock-down of gene expression. We undertook this approach because of the promise of RNAi to easily create cell lines that are specifically deficient in one protein component. By using lentiviral vectors for shRNA expression, high transduction efficiencies can be achieved, avoiding effects due to clonal selection of phenotypically different cells. We successfully targeted the PAI-2 mRNA with the aim of studying the effects of reducing PAI-2 activity on cell sensitivity to apoptosis-inducing stimuli. PAI-2 activity has previously been implicated in protection from apoptosis [ 24 ]. The cytotoxic effect we observed, in cells transduced with RNAi-inducing vectors, appeared to correlate well with the reduction in PAI-2 protein levels. However, rapid selective growth pressure on cells bearing shRNA constructs with scrambled sequences, having no complementarity to the PAI-2 mRNA, suggested non-specific effects rather than a PAI-2-related phenotype. Using GFP as a marker gene, delivered by all vectors, enabled very sensitive detection of selective effects on transduced cells even when initial cell culture suggested stable transduction and cell growth. We were able to detect increased expression of an interferon response gene, OAS1, in cells transduced with all hairpins of 21 or more base-paired nucleotides. A 21 mer hairpin induced the most potent OAS1 induction. These results, and those of others [ 21 , 22 ], suggest that dsRNAs of less than 30 nucleotides can induce an interferon response, even if they cannot directly activate protein kinase R [ 19 ]. The absence of OAS1 induction in cells transduced with 19 mer shRNAs implies that the search for appropriate hairpin sequences should be limited to stretches of this length or less. Overexpression of functional PAI-2 did not rescue the cytotoxic effects or OAS1 induction observed in cells transduced with a series of vectors for expression of different length shRNAs. This result, the cytotoxicity associated with scrambled sequence hairpin-encoding constructs, and the same selective pressure seen on the growth of transduced HeLa cells, which do not express detectable PAI-2 mRNA or protein (Table 2 and data not shown), lead us to conclude that the phenotype we have seen in IS-1 cells is not PAI-2-specific. Without the ability to track transduced cells, via GFP expression, this conclusion would have been more difficult to obtain. In cells engineered to overexpress functional PAI-2, our RNAi-inducing vectors clearly reduced PAI-2 protein levels but did not significantly reduce the overexpressed PAI-2 mRNA. This suggests that in the presence of high concentrations of targeted mRNA, the machinery necessary for RNAi-induced mRNA cleavage is saturated and mRNA down-regulation undetectable. As protein levels are nevertheless reduced, the shRNA may be functioning post-transcriptionally, perhaps in a similar manner to natural miRNA. This phenomenon has been described elsewhere for siRNAs [ 27 ]. IS-1 cell transduction, using one PAI-2 targeting vector (sh119), initially appeared stable, compared to control-transduced cells. PAI-2 protein levels were clearly reduced 10 days after transduction and selective pressure on cell growth appeared to be minimal, as the percentage of sh119 GFP positive cells was apparently stable at about 80 %, 17 days after transduction. However, after cell sorting of GFP positive cells and prolonged cell culture of over one month after transduction, the PAI-2 antigen measured in sh119s (s for selected) cells was restored to control-transduced cell levels. Despite maintenance of transduction marker expression, gene silencing was absent. The GFP expression levels in selected sh119s cells was reduced after one month of growth, compared to cells monitored three days after transduction. It is possible that we selected cells with a greatly reduced, non-cytotoxic shRNA expression level, as we have detected a reduced number of integrated vector copies. The negative selective effect on cells transduced with the sh119 vector was not due to the suppression of PAI-2 expression, as PAI-2 overexpressing cells showed the same negative selection. In all experiments in which a selective pressure on growth was apparent on shRNA-expressing cells, reduced percentage GFP positive cells and reduced GFP expression in transduced cells was measured over time. We hypothesised that very high expression levels of the various shRNAs is cytotoxic. This could occur via high numbers of transcriptionally active vector integration events or integration at transcriptionally active chromosomal regions. Both might be controlled using a tightly regulatable expression system, which has been described [ 15 ], but may require careful dosage in a gene- and cell-specific manner. Our data demonstrate the importance of appropriate controls for using RNAi, as proposed in a recent editorial [ 28 ]. These include suppression of the RNAi phenotype by target gene overexpression, use of scrambled dsRNAs, and monitoring of non-specific gene expression in particular of interferon-responsive genes such as OAS1. Also, if considering the use of stable RNAi, it is imperative that stable knock-down is demonstrated as well as stable marker gene expression. In the cell culture system we used, conclusions drawn from experiments several days after RNAi delivery mask effects which are only apparent days later by monitoring the percentage of transduced cells. Such effects are likely be present in experiments using regulatable RNAi systems or using exogenously added dsRNA, where the experimental data linked to gene targeting may be collected before other effects are seen. The molecular events which lead to the long-term effect we have documented may well be underway during this experimental period. In the light of our own data and other recent reports [ 20 - 22 ], solutions for the induced cytotoxic effects we describe here include testing a series of target sequences, using dsRNA of no more than 19 nucleotides at low effective vector doses, and careful monitoring of transduced cell phenotype with and without functional target gene overexpression. Long-term monitoring of gene silencing appears to be necessary in stable systems, even in the presence of marker gene expression. Conclusions Our study demonstrates vector-derived RNAi in tumor cell lines and points towards the necessity of careful, but clearly feasible, controls when using RNAi for stable gene suppression in short- and long-term experiments. Methods Cell lines Isreco-1 (IS-1) cells were a gift from Dr. B. Sordat (ISREC, Lausanne). 293T cells were a gift from Dr. D. Trono (Geneva University Medical Centre). HeLa cells were purchased from the European collection of cell cultures, ECACC number: 93021013. All cells were maintained in DMEM supplemented with 10 % Fetal Bovine Serum and 10 mM HEPES pH 7.4 (IS-1 medium) (purchased from Invitrogen). Plasmid constructions Gene transfer plasmids, for RNA interference using lentiviral vectors, were constructed using the backbone of ploxEWiresGFP, a gift from Dr. P. Salmon, Geneva University Medical Centre. The human U6 gene was amplified by PCR using HeLa cell genomic DNA as template and the oligonucleotides U6-ClaI-F 5' GATC ATCGATAAGGTCGGGCAGGAAGAGGGCCTATTTCCC 3' and U6-ClaI-R 5' GATCATCGATTGGTAAACCGTGCACCGGCGATAAACG 3'. The 483 base pair PCR product was digested with ClaI, inserted into the ClaI site of pTRE2 hyg (Clontech), and its sequence verified by DNA sequencing. The U6 promoter and gene sequence corresponded to nucleotides 65 to 527 of Genbank accession number M14486. This plasmid was used as template for PCR reactions to amplify U6 promoter-driven expression cassettes. Each PCR product included the U6 promoter with shRNA-encoding sequences beginning at the U6 +1 site, and a run of 6 or 7 thymidine bases for an RNA polymerase III transcription termination signal. Each PCR introduced flanking ClaI restriction sites. PCR products were directly cloned into the pGEM Teasy (Promega) plasmid and sequenced. ClaI fragments of positive clones were excised and ligated into the unique ClaI site of ploxEWiresGFP. The U6 promoter cassettes in the resulting lentiviral vector plasmids were verified by DNA sequencing. Each construct used for vector production had the same orientation of the U6 expression cassette with respect to the P EF-1α-iresGFP region of the plasmid. A schematic of the gene transfer cassette is given in Figure 1A . Details of shRNA sequences used for each construct are given in Table 1 . The gene transfer plasmid for PAI-2 overexpression was constructed by replacing the EGFP gene from ploxCW-GFP (a gift from Dr. P. Salmon, Geneva University Medical Centre) with the type B human PAI-2 (SERPINB2) [ 29 ] open reading frame. Vector production and transduction Lentiviral vectors were produced by three plasmid co-transfection of 293T cells, essentially as described previously [ 30 ]. Vectors were harvested 48 hours after transfection, passed through 0.45 μm filters and used directly on target cells in a 1:1 ratio with IS-1 medium in a total volume of 2 ml. Transductions were performed in 3 cm diameter 6-well plates, on cells seeded the previous day at 5 × 10 4 or 1 × 10 5 cells/well. Upon addition of vectors, plates were centrifuged for one hour at 1000 g in the presence of 8 μg/ml polybrene (hexadimethrine bromide, Sigma). After 24 hours, target cells were washed twice with PBS and cultured in IS-1 medium until analysis. Where indicated, viral titre was determined by transducing cells with 10 μl of lentiviral vector conditioned medium from 293T cell producer cells and measuring the % of GFP positive cells by flow cytometry. We routinely achieve 10 6 effective transducing units per ml of producer cell conditioned medium, which results in a typical multiplicity of transduction of 10. Comparisons between cell populations transduced with different vectors is made by flow cytometry analysis of GFP positive cells. Antibodies Anti-human PAI-2 monoclonal antibody 3750 was purchased from American Diagnostica. PE-labelled goat anti-mouse antibodies were purchased from Pharmingen. HRP-conjugated goat anti-mouse antibodies were purchased from Bio-Rad. Flow cytometry Flow cytometry was performed using Becton Dickinson FACScan, FACStrack or FACScalibur instruments at the Geneva University Medical Centre flow cytometry facility. GFP was detected in cells detached and resuspended in FACS buffer comprising 1 % BSA in PBS supplemented with 0.05 % sodium azide. For the detection of intracellular PAI-2, a method adopted from Dr. M. Ranson (University of Wollongong, Australia) was used. Cells were detached and fixed in 0.25 % paraformaldehyde (PAF)/PBS for one hour on ice. Cells were permeabilised in 0.1 % saponin/PBS for 30 minutes at room temperature. Fixed, permeabilised cells were incubated for 30 minutes in PBS/0.5 % BSA/0.1 % saponin containing 2 μg/ml 3750 anti-PAI-2 monoclonal antibody, washed twice in PBS/0.1 % saponin and incubated for 30 minutes in 0.1 % saponin/0.5 % BSA/PBS/goat anti-mouse-PE antibodies. Finally, cells were washed twice in 0.1% saponin/PBS, twice in PBS and resuspended in 2.5 % PAF for flow cytometric analysis. Sorting of live GFP positive cells was performed using a FACStar+ instrument (Becton Dickinson). Western blotting of cell lysates Cells in suspension were lysed in 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 0.5 % NP-40, 3 mM MgCl 2 , 5 mM DTT and 1 mM PMSF for one hour on ice. Lysates were centrifuged at 16000 g for 5–10 minutes to remove nuclei and precipitates. Supernatant protein concentrations were measured using the Bio-Rad protein assay with BSA in lysis buffer as a standard. Cell lysates were separated by reducing SDS-PAGE and transferred to nitrocellulose membranes. Equal total protein lysate was used for each blot, between 2.5 and 10 μg depending on the assay. Membranes were blocked for 1 hour at room temperature in TBS-0.1 % Tween 20/5 % non-fat milk, and probed using antibodies in TBS-0.1 % Tween 20/5 % non-fat milk. The 3750 anti-PAI-2 antibody was used at a concentration of 1 μg/ml. Microscopy Phase contrast microscopy was performed using a Zeiss Axiovert 100 M instrument on live cells. Images were collected using a Hamamatsu CCD camera (ORCA-100). PAI-2 functional activity To measure PAI-2 functional activity, IS-1 and IS-1 PAI-2 cells were lysed in 1 % NP-40, 150 mM NaCl and 50 mM Tris pH 8.0. Thereafter, 6 μg of cleared total cell lysate was incubated with and without 10 U of low molecular weight urokinase (u-PA). 3 μg total cell lysate samples were then subjected to reducing SDS-PAGE and immunoblotting as for other PAI-2 immunoblots. Quantitative RT-PCR analysis of mRNA Total RNA was isolated from cells using RNA preparation kits from Qiagen or TRIZOL ® reagent (Invitrogen). cDNA was generated using ImpromII ® reverse transcriptase (Promega) and random hexamers, according to the manufacturers instructions, typically using 1 μg of total RNA per reaction. Quantitative PCR was performed using an Applied Biosystems Prism 7000 instrument using Applied Biosystems SYBR ® green master mix reagent and oligonucleotide pairs to detect hypoxanthine phosphoribosyl transferase (HPRT), PAI-2 and oligoadenylate synthase-1 (OAS1) cDNA. 5' to 3' primer sequences were as follows: HPRT forward TATTGTAATGACCAGTCAACAG, HPRT reverse GGTCCTTTTCACCA GCAAG, PAI-2 forward GGGTCAAGACTCAAACCAAAG, PAI-2 reverse CCTTTGAAGTAGACAGCATTC, OAS1 forward AGGTGGTAAAGGGT GGCTCC and OAS1 reverse ACAACCAGGTCAGCGTCAGAT. Data were analysed using Applied Biosystems Prism software and the ΔC T method. Briefly, target gene expression was normalised to the HPRT endogenous reference gene for each sample. The difference between mean threshold PCR cycle values for target and control genes gave the ΔC T value. This was then calibrated to the control sample in each experiment to give the ΔΔC T value, where the control had a ΔΔC T value of 0. The fold target gene expression, compared to the calibrator value, is given by the formula 2 -ΔΔCT . Error bars represent the standard deviation of each target gene value, after evaluating the expression 2 -ΔΔCT+s and 2 -ΔΔCT-s , where s is the standard deviation of the ΔΔC T value. All reactions were performed in duplicates or triplicates. ShRNA detection Expression of shRNAs was detected using the mir Vana™ Probe Construction and miRNA Detection kits from Ambion (Austin), according to the manufacturers instructions. These kits employ in vitro transcription for radiolabelled probe generation and an RNase protection protocol for detection of small RNA expression, respectively. Briefly, radiolabelled RNA probes incorporating α- 32 P-UTP (Amersham) were constructed using T7 polymerase-driven transcription templates. Templates were designed to generate RNAs which hybridize to the endogenous Mir-16 miRNA, as a control, or the first 19 nucleotides of the sh325 shRNA (see table 1 ). The sh3 probe should therefore hybridize to any of the sh319/sh321/sh323 or sh325-derived shRNAs. Before hybridization, radiolabelled probes were purified by migration of the transcription reaction on a 12 or 15% Acrylamide/8 M Urea denaturing gel and elution of radioactive probe bands after detection by autoradiography. 3 μg of total cellular RNA was used for all hybridizations and RNase protections, supplemented with 2 μg yeast RNA as a carrier. Control reactions without target RNA but with or without RNase digestion included 5 μg yeast RNA. After hybridization and RNase digestion, protected probes were detected by autoradiography after migration in a 15% acrylamide/8 M Urea denaturing gel. Authors' contributions RF carried out all of the experiments in this study and contributed to its conception, design and description. EKOK conceived the study and participated in its design and description. Both authors approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514603.xml |
521684 | Strengths and weaknesses of EST-based prediction of tissue-specific alternative splicing | Background Alternative splicing contributes significantly to the complexity of the human transcriptome and proteome. Computational prediction of alternative splice isoforms are usually based on EST sequences that also allow to approximate the expression pattern of the related transcripts. However, the limited number of tissues represented in the EST data as well as the different cDNA construction protocols may influence the predictive capacity of ESTs to unravel tissue-specifically expressed transcripts. Methods We predict tissue and tumor specific splice isoforms based on the genomic mapping (SpliceNest) of the EST consensus sequences and library annotation provided in the GeneNest database. We further ascertain the potentially rare tissue specific transcripts as the ones represented only by ESTs derived from normalized libraries. A subset of the predicted tissue and tumor specific isoforms are then validated via RT-PCR experiments over a spectrum of 40 tissue types. Results Our strategy revealed 427 genes with at least one tissue specific transcript as well as 1120 genes showing tumor specific isoforms. While our experimental evaluation of computationally predicted tissue-specific isoforms revealed a high success rate in confirming the expression of these isoforms in the respective tissue, the strategy frequently failed to detect the expected restricted expression pattern. The analysis of putative lowly expressed transcripts using normalized cDNA libraries suggests that our ability to detect tissue-specific isoforms strongly depends on the expression level of the respective transcript as well as on the sensitivity of the experimental methods. Especially splice isoforms predicted to be disease-specific tend to represent transcripts that are expressed in a set of healthy tissues rather than novel isoforms. Conclusions We propose to combine the computational prediction of alternative splice isoforms with experimental validation for efficient delineation of an accurate set of tissue-specific transcripts. | Background The large difference between cells from different tissues is the consequence of a complex regulatory machinery guiding the tissue specific expression of genes and their transcripts. Several genes have been described to exhibit differential splicing patterns for different tissues (E.g. PDE1C [ 1 ]; IRF-3 [ 2 ]) that result either in alternative proteins or affect the regulation of the respective gene product [ 3 ]. Due to the large number of genes generating alternative transcripts as well as by the complicated splicing machinery involving a large variety of different proteins, mis-splicing events are also frequently observed. Some of these artificial splice isoforms are already linked to specific diseases like Hemophilia A, Marfan syndrome etc. [ 4 , 5 ]. The resource mainly used to predict tissue-specific expression is the rapidly expanding repertoire of expressed sequence tags (ESTs) in the public databases representing a wide spectrum of tissue types. Unlike serial analysis of gene expression (SAGE) which mainly yields the tissue specific expression of genes [ 6 ], the EST data additionally allow the identification of alternatively spliced transcripts [ 7 - 11 ]. Besides the detection of the existence of alternative splice isoforms the tissue annotation of ESTs can be used for the computational prediction of the expression pattern of these transcripts where the tissue-wise count of transcript-specific ESTs with respect to a random background distribution defines an expression level [ 12 - 14 ]. Transcripts that are significantly over-represented by ESTs derived from a single tissue are usually defined as being tissue-specifically expressed. However, different cDNA construction protocols like normalization [ 15 ] include subtractive hybridization and PCR amplification steps introducing an artificial enrichment of ESTs from lowly abundant transcripts. The level of enrichment depends on the number of normalization/amplification steps performed, measured as Cot or Rot [ 16 ]. This inconsistency in the correlation of the number of ESTs observed for a transcript and its real expression level may affect the reliability of computational predictions of tissue-specifically expressed transcript. Since the EST-based prediction of expression patterns might already be error-prone because of the lack of sufficient numbers of EST sequences for each tissue this might be further complicated by different cDNA library protocols. Consequently, EST data related to normalized cDNA libraries are excluded from analysis in several computational approaches that aim at predicting tissue-specific expression [ 13 , 17 ]. Because of these uncertainties we combined our computational prediction of alternative splice variants and their expression pattern with experimental validation of these iso-forms via RT-PCR on 40 different tissue samples in order to evaluate the predictive potential of ESTs. Results The EST-based prediction of alternative splice iso-forms revealed 427 genes each contributing at least one potential tissue-specifically expressed variant. These variants show specificity for 28 different tissue types, where brain, testis and placenta account for approximately half of these transcripts (see additional file 1 ). Many of these genes (n = 210) exhibit isoforms that were exclusively detected due to ESTs derived from normalized libraries. These form a significant fraction (p-value: 8e-19) of the total genes that show tissue specific transcripts, since the number of ESTs derived from normalized libraries (896,645) is only 30% the total EST count (3,084,576) in tissues for which tissue specific isoforms exist. Out of the 20 isoforms tested experimentally (see additional file 3 for details of experiments), 15 isoforms could be successfully verified in some tissue (Table 1 ). The remaining five variants are either likely to resemble rare transcripts according to the respective library construction protocol, or as in case of a disease-specific isoform (Hs.272688), the appropriate tissue sample was not available for experimental testing. Only four of the isoforms predicted based on the basis of normalized libraries could be validated using the standard RT-PCR conditions. For five additional isoforms a more refined protocol had to be applied in order to detect bands of significant strength. More sensitive PCR conditions frequently revealed expression in more tissues indicating low expression of the isoforms in these tissues. These results show the tendency of normalized libraries to be enriched for low-abundant transcripts. Table 1 RT-PCR validation results for tissue and disease-specific splice isoforms. The experiments are categorized into three groups viz. tissue specific isoforms predicted via ESTs related to non-normalized libraries (1 to 4), tissue specific isoforms predicted only via ESTs derived from normalized libraries (5 to 16) and disease-specific isoforms (17 to 20). For some of the variants represented by normalized libraries, standard PCR did not reveal the isoforms. However, five of these isoforms were detected using refined PCR conditions. The experiments frequently validated the isoforms and the tissue type, but the predicted specificity was rarely verified. Isoform Gene Chr. Unigene EST Evidence ESTs Cycles Isoform Specificity Comment (Most sensitive PCR) Norm. Level 1 Unknown 11 Hs.112250 testis 3 39 + + 2 ISGF3G 14 Hs.1706 stomach 10 39 + - Ubiquitous 3 MRPL42 12 Hs.112110 stomach-lymph 5 39 + - Ubiquitous 4 SGN3 17 Hs.6076 testis 3 39 - ? 5 PC326 13 Hs.279882 testis 9 39 + + testis [36] Rot-5 6 LMO7 13 Hs.5978 brain 5 39 + - brain, testis, eye(?) [18] 7 HRD1 8 Hs.274122 brain 3 39 + - brain, eye, thymus, salivary gland, kidney 8 Unknown 1 Hs.24119 pancreas 4 39 + - approx. 10 tissues Cot-20 9 BCLG 12 Hs.11962 testis 4 39,78 ?,+ +,+ Cot-5 10 RBPMS 8 Hs.80248 placenta 4 39,78 ?,+ -,- Ubiquitous 11 SCML1 X Hs.109655 testis 12 39,78 ?,+ +,- approx. 6 tissues Rot-5 12 WNK1 12 Hs.432900 kidney 3 39,78 ?,+ +,- Digestive system [28] Cot-25 13 NY-CO-10 5 Hs.23557 testis 3 39,78 -,+ ?,+ Cot-5 14 Unknown 11 Hs.169100 testis 3 39,78 -,- ?,? Rot-5 15 Unknown 16 Hs.48396 breast 4 39,78 -,- ?,? Cot-230 16 CIDE-A 18 Hs.249129 breast 4 39,78 -,- ?,? Cot-230 17 KCNAB2 1 Hs.298184 tumor 29 39 + - Ubiquitous 18 SNRP70 19 Hs.174051 stomach ascites 25/26 39 + - Ubiquitous 19 RAB1 14 Hs.227327 tumor 39/95 39 + - fetal(kidney, thymus, liver, spleen), ovary [19] 20 Unknown 7 Hs.272688 tumor 12 39,78 -,- ?,? relevant tumor sample not in set The predicted expression of the isoforms in a single tissue could not be confirmed for half of the variants analyzed (standard conditions). However, the isoforms were always detected to be expressed in the tissue that was originally predicted by our software. The observed expression pattern of the 'unspecific' isoforms ranges from expression in only a few, sometimes related tissues (LMO7 [ 18 ]: brain, eye, testis, Fig. 2 ; HRD1: brain, eye, thymus, salivary gland, kidney) to ubiquitous expression (MRPL42, ISGF3G). Those variants that were validated to be specifically expressed frequently originate from testis. Increasing the sensitivity of the RT-PCR revealed another testis-specific variant. At the same time the variants of the genes WNK1 and SCML1 were no longer defined as being tissue-specifically expressed since they were now also detected in a few additional tissues (Table 1 : isoform 11 & 12). The number of genes with transcripts exclusively expressed in tumors is relatively large (1120) as compared to the number of genes revealing tissue specific isoforms. Interestingly, 2 out of 4 such disease-related transcripts (Table 1 : isoform 17–20) were ubiquitously expressed although the large number of ESTs covering these variants was suggesting a high significance of the prediction. The tumor associated isoform described by Wang et al. [ 19 ] was observed to be expressed in several fetal tissues along with ovary. Figure 2 RT-PCR validation experiment of a putative brain-specific isoform ( LMO7 ) . (A) The additional exon is detected in all tissues (primers F1, R1). (B) The primer pair F1-R2 located on exons flanking the extra exon results in two products where the shorter one is observed in brain, testis and eye (weak band). The predicted brain-specific expression pattern is, in fact, not specific. The entire dataset for human as well as the gel images from the RT-PCR experiments is available at . Discussion Consistent with previous work [ 11 ] our approach of combining computational and experimental validation yields a high success rate in predicting the existence of splice variants. In line with the expected general enrichment of clones derived from lowly expressed transcripts in normalized cDNA libraries our experimental results confirm the expression of the predicted low abundance transcripts. Consequently, those isoforms that could not be validated experimentally may also reflect real biological signatures of extremely rare transcripts since they are often represented just by heavily normalized libraries (Cot 230, CIDE-A + Hs.48396). While the methods used in the construction of normalized libraries (PCR amplification, subtraction, size selection) increase the sensitivity of the detection of transcripts they unfortunately disturb the rough correlation between the expression level of a transcript and the observed number of related clones that is usually maintained in non-normalized libraries. Therefore, in these cases, the larger number of ESTs found for a specific transcript will profess to deal with a higher expressed transcript, also implying a higher confidence in the prediction although the sequences may be derived from the same although amplified clone. From the computational point of view the artificially increased number of ESTs affects the likelihood to predict tissue-specifically expressed transcripts since the prediction mainly relies on the count of ESTs [ 12 , 13 ]. Nevertheless, our experimental results show that especially isoforms predicted to be expressed exclusively in testis could be successfully validated, while other isoforms frequently appear to be expressed in a set of additional tissues that were not suggested by the ESTs. Surprisingly, the absence of supporting EST evidence for the variants LMO7 and ISGF3G is not caused by the lack of the respective cDNA libraries but may rather reflect differences in the tissue samples (e.g. enrichment of different cell types from the same organ, developmental differences) used for library construction. In the context of tumors, our data shows that the predicted tumor-specific expression of isoforms derived from ESTs usually tends not to reflect the experimentally validated expression pattern. Rather it suggests expression in a collection of different tissues although the large number of related ESTs derived from tumor would imply a high confidence in the EST based prediction. Since tumor cells often show an up-regulation of a larger number of transcripts involved in various pathways [ 20 , 21 ] the tumor-specific transcripts predicted based on the EST data may just reflect this general deregulation of gene expression. The large number of predicted tumor-related isoforms further supports this hypothesis. Nevertheless, some transcripts detected via EST data may still serve as potential tumor markers like in case of the gene PRAME [ 22 ] where the EST data as well as the experimental data suggests specific expression in testis and in a variety of different tumors (see additional file 2 ). Overall, ESTs are an extremely powerful tool to reliably unravel alternative transcripts independent of the level of expression. The functional relevance of the low abundant transcripts is not yet clear, especially if the isoforms do not affect the coding sequence. These isoforms may either be related to processes like nonsense-mediated decay (NMD: [ 23 , 24 ]) or they might be some kind of non-functional leakage of the splicing machinery. Nevertheless, since many lowly expressed genes are already known to have important regulatory functions [ 25 - 27 ] this may also hold true for a not yet defined fraction of the alternative isoforms we detected via normalized libraries. In contrast to the prediction of the existence of isoforms, the task of predicting their expression pattern is much more error-prone since EST data always covers only a subset of potential tissues with variable sensitivity. The fuzzy terminology of tissue-specific expression that is frequently used to describe significant expression in a discrete tissue or a set of tissues, is therefore strongly biased by the sensitivity of computational and experimental methods (SCML1; WNK1 [ 28 ]). Beside these technical difficulties, the definition of specificity may also depend on the regulatory network that mediates tissue-specificity. While isoforms expressed in testis are specifically expressed in a more strict sense, other isoforms are expressed in a small set of (not necessarily related) tissues eventually pointing to alternative regulatory mechanisms acting with different stringency, e.g. involving transcription factors [ 29 ], [ 30 ] and/or DNA methylation [ 31 , 32 ]. Conclusions The separate evaluation of EST data from non-normalized as well as from normalized cDNA libraries will help to categorize transcripts into highly and lowly abundant ones thus facilitating the integration of EST-based predictions with expression data from microarray experiments. We suggest that large-scale analysis of tissue-specific transcripts should be ideally based on a computational prediction of isoforms that ranks candidate transcripts, tightly coupled with experimental validation via RT-PCR or DNA microarray experiments [ 33 ]. Such an approach will lead to a comprehensive set of verified isoforms suitable for a wide range of applications in the functional analysis of the regulation of tissue-specific expression. This will also improve the detection of tumor-related isoforms that do not just reflect a general up-regulation of gene expression. Methods The basis of our work is the tissue/tumor annotation of ESTs is GeneNest database [ 34 ] and the quality prediction of alternative splicing [ 11 ], visualized in SpliceNest database [ 10 ]. Library classification The cDNA libraries of the GeneNest database were semi-automatically categorized into non-normalized, normalized/subtracted and PCR-based libraries by screening for the appropriate keywords in the original annotation of the respective EMBL database entries. All libraries for which none of the keywords were found were defined as being non-normalized. PCR-based libraries like those derived by ORESTES PCR were not used for the current analysis. Additionally, to avoid miscounting caused by PCR amplification, ESTs of the same library and with identical start/end positions in the alignment were treated as a single sequence. Since the level of normalization of different libraries may differ depending on the number of rounds of subtractive hybridizations performed, we also extracted the normalization level (measured as Cot or Rot: [ 16 ]) as far as it was noted in the respective entries. Increasing Cot-values hereby reflect the enrichment of clones derived from low abundant transcripts in the respective cDNA library. Besides the categorization of cDNA libraries according to the construction methods used we further split these groups into libraries derived from healthy or disease tissue. Finally, ESTs of the four groups of cDNA libraries (healthy/non-normalized, healthy/normalized, disease/non-normalized, disease/normalized) were either analyzed separately or data of normalized and non-normalied libraries were combined. Prediction of tissue specific alternative splicing Alternative splice isoforms in the SpliceNest database are revealed by aligning EST consensus sequences (putative transcripts) related to one gene to the appropriate genomic sequence. Significant differences in the boundaries of the putative exons are interpreted as alternative splicing events. For all exon-exon-boundaries that define a certain splice iso-form the annotation of ESTs covering the respective boundary is evaluated. Isoforms overrepresented by ESTs from particular tissue are tagged as putative tissue/tumor specific splice isoforms. Several parameters (e.g. number of ESTs from a particular tissue, number of ESTs from other tissues, number of associated mRNA sequences etc.) are computed for these isoforms and finally stored in a relational database system. The refined set of tissue and tumor specific variants is then generated by setting the requirement of at least 3 ESTs in both alternative forms. Fig. 1 describes such a prediction using GeneNest and SpliceNest visualizations. Since the counts of ESTs per tissue-specific splice event were frequently below 5, we considered it inappropriate to apply statistical methods as were used by Xu et. al. ([ 12 ]). Figure 1 Detection of brain specific splicing in gene LMO7 . The top part of the figure is a visualization of gene LMO7 in SpliceNest, showing parts of three transcripts with exons displayed as red blocks, connected by lines representing introns. The middle exon of the top transcript (Hs5978.1) is missing in the second transcript (Hs5978.2) and is therefore highlighted as an alternative splice event (green bar). The boundaries corresponding to this exon as well as the corresponding intron are visualized as vertical lines in the GeneNest database (left and right box respectively). Both regions are covered by several ESTs depicted by horizontal arrows with corresponding tissues encoded in coloured rectangles towards the left of each EST. Upon comparing the tissue distribution of these alternative regions it is evident that the middle exon of transcript Hs5978.1 is covered by ESTs derived from several tissues, while the corresponding exon junction that lacks this middle exon, in transcript Hs5978.2, is represented by ESTs derived from brain only, thereby revealing this as a brain specific splice event. Experimental verification A set of putative tissue specific (n = 16) and disease-related (n = 4) alternative splice events was arbitrarily selected for RT-PCR experiments. PCR primers were generated on the alternatively spliced exon as well as on either side of the event (Fig. 2 ) using the primer design software GenomePRIDE ([ 35 ]). For the subsequent RT-PCR experiment, total RNA was prepared using the single-step guanidinum method according to the manufacturer's instructions (TRIZOL, Gibco BRL). First strand cDNA synthesis was carried out in 20 μl reaction using the Omniscript Reverse transcriptase (Qiagen) and the oligo(dT) primers with 2 μg of total RNA. RT-PCR was carried out in a 20 μl reaction in 1 × buffer [1.5 mM Mg2+, 0.2 mM dNTPs, 0,4 μM primers each, 1 Unit of Taq polymerase (Roche)] and 1 μl of cDNA. Amplification steps were as follows: 95°C for 90 sec; 9 cycles of 94°C for 20 sec, 64°C for 10 sec decreasing the annealing temp for 1°C with each cycle (touchdown), 72°C for 20 sec; followed by 30 cycles of 94°C for 20 sec, 55°C for 10 sec, 72°C for 20 sec, followed by an extension at 72°C for 10 min. For the refined PCR, the amplification step was repeated with identical PCR conditions but with 2 μl of PCR product instead of 1 μl of cDNA. All PCR products were resolved on 2% agarose gels run at 90 V/20 cm for 1.5 h in TAE buffer. Gels were then manually examined for exact size, genomic contamination and the tissues in which the bands are observed. As a control, a fraction of variants were sequenced using the ABI Prism BigDye Terminators and the ABI Prism 3100 sequencer (Applied Biosystems). Authors' contributions SG wrote the prediction software as well as designed PCR primers for experimental analysis. SH and MV provided guidance for the computational work. DZ performed the RT-PCR experiments with the guidance of BK. Supplementary Material Additional File 1 List of tissues for which tissue specific transcripts are predicted. This is a text file with a listing of all tissues for which specific trascripts exist along with the number of ESTs related to individual tissues. Furthermore, the ESTs derived from normalized libraries and the specific variants predicted via such ESTs are also listed. Click here for file Additional File 3 Detailed description of RT-PCR experiments This is an excel file containing the primer sequences used for RT-PCR experiments along with detailed comments on the gel pictures subsequently obtained. Click here for file Additional File 2 RT-PCR picture (jpeg file) showing the expression pattern of gene PRAME This gene shows specific expression for several tumor types, along with testis as the only normal tissue. Click here for file | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521684.xml |
546186 | Differences in codon bias cannot explain differences in translational power among microbes | Background Translational power is the cellular rate of protein synthesis normalized to the biomass invested in translational machinery. Published data suggest a previously unrecognized pattern: translational power is higher among rapidly growing microbes, and lower among slowly growing microbes. One factor known to affect translational power is biased use of synonymous codons. The correlation within an organism between expression level and degree of codon bias among genes of Escherichia coli and other bacteria capable of rapid growth is commonly attributed to selection for high translational power. Conversely, the absence of such a correlation in some slowly growing microbes has been interpreted as the absence of selection for translational power. Because codon bias caused by translational selection varies between rapidly growing and slowly growing microbes, we investigated whether observed differences in translational power among microbes could be explained entirely by differences in the degree of codon bias. Although the data are not available to estimate the effect of codon bias in other species, we developed an empirically-based mathematical model to compare the translation rate of E. coli to the translation rate of a hypothetical strain which differs from E. coli only by lacking codon bias. Results Our reanalysis of data from the scientific literature suggests that translational power can differ by a factor of 5 or more between E. coli and slowly growing microbial species. Using empirical codon-specific in vivo translation rates for 29 codons, and several scenarios for extrapolating from these data to estimates over all codons, we find that codon bias cannot account for more than a doubling of the translation rate in E. coli , even with unrealistic simplifying assumptions that exaggerate the effect of codon bias. With more realistic assumptions, our best estimate is that codon bias accelerates translation in E. coli by no more than 60% in comparison to microbes with very little codon bias. Conclusions While codon bias confers a substantial benefit of faster translation and hence greater translational power, the magnitude of this effect is insufficient to explain observed differences in translational power among bacterial and archaeal species, particularly the differences between slowly growing and rapidly growing species. Hence, large differences in translational power suggest that the translational apparatus itself differs among microbes in ways that influence translational performance. | Background Translational power is the rate of protein synthesis of a cell or culture, normalized to the amount of biomass invested in the protein synthesis machinery. We are introducing the term 'translational power' to describe precisely the same concept (and the same quantitative parameter, see Methods) that was originally defined as 'ribosome efficiency' [ 1 - 3 ]. In recent years, this concept has more commonly been called 'translational efficiency' [ 4 , 5 ], particularly in discussions of codon usage bias [ 6 - 8 ]. Although we are reluctant to depart from established terminology, we do so to avoid an inconsistency with the meaning of 'efficiency' as it is used in many other areas of science and in common parlance. In the physical sciences and in many areas of biology, the efficiency of a process refers to a comparison of output to input, in particular to the fluxes of useful energy and/or mass (e.g., the efficiency of a heat engine [ 9 ], trophic transfer efficiency [ 10 ]). These scientific meanings of 'efficiency' are consistent with the common notion that a process obtaining the desired output with little waste is highly efficient. According to these conventions, calculations of efficiency make no direct reference to the rate at which a process occurs. Physicists and engineers use a distinct term, 'power,' to refer to the rate of energy consumption or the rate at which work is performed [ 11 ]. The semantic distinction between power (or rate) and efficiency is important, because in many real and idealized physical systems, the laws of thermodynamic result in an inherent tradeoff between power and efficiency [ 9 ]. In biology, several attempts to argue for the universality of power-efficiency tradeoffs [ 12 , 13 ] have justifiably been criticized for the misapplication of thermodynamic arguments [ 14 - 16 ]. Nonetheless, many specific tradeoffs have been demonstrated in a wide range of organisms that can be described as evolutionary choices between power (increased rates of biological processes such as resource acquisition, metabolism or organismal growth) and efficiency (increased biological output measured as probability of survival, production of biomass, number of progeny, etc. per unit resource) [ 17 - 24 ]. Among bacteria, comparisons of coexisting species or strains have also provided evidence for power-efficiency tradeoffs [ 25 - 28 ], as have comparisons of engineered mutant strains [ 29 , 30 ]. However, the absence of apparent tradeoffs in some carefully designed studies of bacteria demonstrates that such tradeoffs are not inevitable [ 31 - 33 ]. Even if power-efficiency tradeoffs occur only in some biological contexts, it is valuable to maintain a semantic distinction between power (implying rapid rate) and efficiency (implying low waste). However, the terms 'ribosome efficiency' and 'translational efficiency' blur this distinction, because they refer to a rate – the quantitative measure of ribosome efficiency [ 1 ] is expressed in units of (time -1 ). We prefer the term 'translational power', which refers to the rate of protein synthesis of a cell or culture, normalized to the mass of the translational apparatus, in a manner that is more consistent with the connotations of 'power' and 'efficiency' derived from other areas of science and from colloquial usage. Translation rate (a synonym of 'protein chain growth rate' [ 3 , 34 ], meaning the rate of amino acid polymerization per active ribosome) is one component of translational power, but translational power reflects other properties of the protein synthesis system as well, most notably the fraction of ribosomes that are active (see Methods, also chapter 6 of reference [ 34 ]). Intuitively, translational power measures the capacity of the protein synthesis subsystem to drive replication of the cell, the protein-dominated autocatalytic system to which it belongs. The concept and a quantitative metric of translational power were first introduced to facilitate comparisons of translational performance between different growth rates within a single bacterial strain [ 1 ]. The initial belief that translational power is nearly constant in a strain across a wide range of growth rates, based both on empirical data and theoretical arguments [ 2 , 34 ], has gradually given way to the current understanding that translational power increases with growth rate, at least in E. coli [ 3 , 4 , 35 , 36 ]. The question of whether translational power varies between microbial species has been investigated only rarely, in four studies that each compared a single slowly-growing microbial species to E. coli [ 37 - 40 ]. In each case, translational power was found to be higher in E. coli than in the slowly growing comparison strain. Although each of these studies discusses this unexpected result, only one of them references the same result from another study. In previous work, the consistent association of low translational power with slowly growing microbes appears to have escaped notice; however, our reanalysis of the data from these four studies as well as additional published data (presented in Results) suggests that the association is robust. One factor capable of affecting translational power is the biased usage of synonymous alternative codons. In the standard translational code, 18 of the 20 amino acids are encoded by more than a single codon, but in many microorganisms, synonymous codons are not used with equal frequency. The pattern first found in E. coli and Bacillus subtilis turns out to be common: the majority of genes within an organism show a preference for the same subset of codons, but the degree of bias towards the preferred subset is correlated with the expression level of the gene [ 41 , 42 ]. For some time, the consensus has been that such a pattern reflects selection for translational power [ 7 , 8 ]. Codon bias increases translational power because preferred codons tend to be translated more rapidly than synonymous alternatives [ 43 - 45 ]. This effect can be attributed to the high abundance of tRNAs cognate to the preferred codons, to a canonical base pair interaction at the codon wobble position between preferred codons and their cognate tRNAs, or to both these factors [ 7 , 8 ]. Codon bias resulting from selection for translational power (or for any other translation-dependent benefit) is correlated with gene expression level because the benefit accrues during each instance of translation, so the selective pressure for preferred codons is stronger in more highly expressed genes [ 7 , 8 ]. In contrast to the codon bias caused by translational selection, codon bias that is consistent in both magnitude and direction in genes that vary widely in expression level is explained most easily by mutational bias acting on DNA [ 8 , 46 ]. While the effects of both translational selection and mutational bias are evident in some microbial genomes with moderately biased G+C content [ 47 , 48 ], organisms with strong mutational bias (very high or low G+C content) have been reported to show very little [ 49 ] or no [ 50 - 52 ] evidence of translational selection. Theoretical calculations indicate that if the strength of mutational bias exceeds a certain critical threshold, any pre-existing codon preferences that conflict with the mutational bias will be reversed [ 53 ]. In this case, codon use is almost entirely determined by the mutational bias, which influences genes equally regardless of expression level. Note that while the degree of codon bias and the gene expression level would not be correlated among genes from such a genome, this does not necessarily imply that deviations from the average (biased) codon usage would be selectively neutral, nor that the fitness effects of any such deviations would be independent of gene expression level. The absence of a correlation between codon usage and gene expression level has also been reported in some organisms with moderate G+C content, in particular the spirochete Treponema pallidum [ 54 ] and the proteobacteria Helicobacter pylori [ 55 ]. The lack of evidence for translational selection in these organisms requires an explanation, since they lack a strong mutational bias that could obscure such evidence. It has been suggested that rapid exponential growth confers little or no fitness benefit in these strains [ 8 , 55 ], consistent with their slow growth rate and other characteristics of their ecological niche. If so, these organisms would not experience selection for translational power. If variation in the strength of selection for translational power leads to differences in the degree of codon bias between microbes (superimposed on any differences in codon bias that can be attributed to variation in mutational bias), we wondered whether differences in codon bias could in turn explain the observed differences in translational power between microbes. An estimate of the effect of biased codon use on the overall rate of translation would depend on knowledge of absolute or relative translation rates in vivo for each codon. Unfortunately, these data are incomplete even for E. coli , and are not available for other microbes. Therefore, we approach the issue by framing the following question: How much faster is the translation rate of E. coli than the expected translation rate of a hypothetical organism that has the same proteome composition and the same investment in translational machinery as E. coli , but which lacks codon bias? Here we report results from a simple mathematical model developed to address this question. For convenience, we will refer to the hypothetical E. coli -like organism with uniform use of synonymous alternative codons as 'Uni'. By 'same proteome composition', we mean that over a cell generation, each amino acid is incorporated into protein the same number of times in Uni and in E. coli , although for the 18 amino acids specified by multiple codons, the individual codons will differ in frequency. By 'same investment in translational machinery', we mean that the total biomass of the translational apparatus is the same in Uni and in E. coli , although ideally the allocation of that biomass among various components of the apparatus in Uni would be optimized for unbiased codon usage. However, in order to apply empirical codon-specific translation rate data, we will impose a more stringent requirement on Uni, that the abundance of each individual component of the translational apparatus will be unchanged in comparison to E. coli . Due to this restriction, and due to the incomplete data for codon-specific translation rates, we make no claim to be able to answer our question precisely. However, our approximations are adequate to conclude that differences in codon bias alone are unlikely to account for differences in translational power of the magnitude inferred from macromolecular analysis of slowly growing and rapidly growing microbes. Results Comparisons of translational power among microbes We know of 4 studies that have made explicit comparisons of translational power between different microbial species; in each case, the comparison was made between E. coli and a single slowly growing strain [ 37 - 40 ]. One of these studies relied on original measurements of E. coli [ 38 ]; the remaining studies made comparisons using the E. coli data of Bremer and Dennis [ 3 ]. Although growth rates and translation rates vary with temperature [ 56 ], at least 2 of the 4 studies [ 39 , 40 ] compared data from strains grown at different temperatures without compensating for temperature effects. One of 2 studies that made comparisons based on the number of ribosomes per cell volume appears to have assumed that E. coli cell volume is constant over a range of growth rates [ 39 ], which is unlikely. We have reanalyzed the data from these studies (as described in Methods) to provide consistent comparisons of translational power between E. coli and other strains. In addition, we applied the same comparative methodology to every microbial species for which we could find the requisite data in the literature. The list of species that could be included is surprisingly short; most studies reporting both the protein and RNA content of microbes growing at known rates have involved E. coli or closely related enteric bacteria. Table 1 summarizes the comparisons of translational power between E. coli and all other species. The comparisons of translational power in Table 1 are based on the fastest growth rate for which data are available for each of the comparison organisms, because at submaximal growth rates, there may be a reduction in the average translation rate [ 4 , 57 ], in the active fraction of ribosomes [ 35 , 36 ], or both. Either of these phenomena would reduce translational power. However, the comparisons to E. coli are not always based the fastest E. coli growth rate, but rather on the growth rate at which E. coli makes a comparable investment in the translational apparatus as the comparison organism. A comparison at similar investment levels reflects the expectation that the selective pressure to maximize translational power increases with the biomass invested in the apparatus [ 4 , 58 ]. If the comparisons had always been made to the fastest E. coli growth rate (i.e., where E. coli translational power is highest), the disparity in translational power would be greater for most of the comparisons shown. Even with the conservative comparisons displayed in Table 1 , the published data suggest that translational power varies considerably between strains, particularly for comparisons between microbes adapted to different ranges of growth rates. While translational power is higher in E. coli and other rapidly growing organisms, it is lower in slowly growing organisms, ranging from less than 17% to 42% of the value for E. coli . Hence, if differences in the degree of codon bias are to explain these differences in translational power, we would expect codon bias to be capable of accelerating the rate of translation by 2.5-fold to 6-fold. In summarizing the comparisons of Table 1 as a contrast between slowly growing and rapidly growing microbes, we are not relying on the actual growth rates shown in the third column, especially since chemostat growth rates are necessarily constrained below the maximal growth rate for a strain. Instead, we have relied both on well-recognized growth characteristics for some species (e.g., Sphingopyxis alaskensis and Rickettsia prowazekii are slow growers, Salmonella enterica and Enterobacter aerogenes are rapid growers), and on the number of copies of the ribosomal RNA ( rrn ) operon per genome. High rrn copy number is an adaptation permitting rapid growth [ 59 , 60 ], while low rrn copy number is characteristic of microbes adapted for slow growth [ 39 , 61 ]. Estimates of the translation rate benefit of codon bias We define the translation rate benefit of codon bias in E. coli as s bias , the fractional increase in the time required to replicate the E. coli proteome if the actual codon bias of E. coli were to be replaced with uniform use of synonymous codons (Equation (10) in Methods). Our estimates of s bias depend on the relative translation rates of individual codons in vivo , and on the frequency with which each codon is used in synthesizing the proteome. The sources we have used for these data, and the details of several adjustments made to the source data, are described in the Methods section. All data used in our estimates of s bias are presented in Table 2 . Because the codon-specific translation rate data are incomplete even for E. coli , we have explored 4 different scenarios (described in Methods) for extrapolating from the empirical rate data to obtain an estimate of s bias over all codons. Scenarios 1–4 are increasingly complex, and represent deliberate attempts to assign translation rates to the unmeasured codons in a way that increases s bias while remaining consistent with patterns found in the empirical data. Furthermore, in Scenario 5, we apply a theoretical approach [ 62 ] for predicting optimal codon-specific translation rates that does not rely on empirical translation rate measurements at all, but only on codon frequency and tRNA abundance data. Estimates of s bias for all scenarios are presented in Figure 1 . The empirical translation rate data used in Scenarios 1–4 reflect ternary complex selection at the ribosomal A-site, but not translocation of the newly-formed peptidyl-tRNA from the A-site to the P-site [ 45 ]. Thus, for these scenarios we show two estimates of s bias that are based on different assumptions regarding the relative duration of translocation and ternary complex selection. The white bars of Figure 1 are based on the assumption that the duration of translocation is negligible for all codons in comparison to the duration of ternary complex selection. The cross-hatched bars of Figure 1 are based on the assumption that translocation requires a finite amount of time that is constant for all codons, but short in comparison to the time required for ternary complex selection [ 63 ]. In Scenario 5 the duration of translocation is not treated explicitly, but the theoretical rate predictions refer to the entire cycle of translational elongation. Hence, we have grouped the estimate from Scenario 5 with other estimates that account for the duration of translocation. Our estimates of the benefit of codon bias in E. coli relative to the complete absence of codon bias range from 0.6 – 1.4 if translocation time is neglected, or from 0.4 – 1.1 with the more realistic assumption that translocation requires a short amount of time. We have also estimated the benefit of codon bias in E. coli relative to the limited degree of codon bias that might be found in an actual low-bias organism, rather than making a comparison to the biologically unrealistic standard of strictly uniform synonymous codon use. We took T. pallidum as our example of a microbe with limited codon bias, since it is a slowly growing bacterium with little mutational bias (52.7% G+C) that has also been reported to lack translational selection [ 54 ]. The T. pallidum genome has the second-most uniform codon use over all predicted genes (assessed as Wright's effective number of codons [ 64 ]) among 108 bacterial and archaeal species for which complete genome sequences were available in June, 2003 (data not shown). Our method for generating a set of low bias codon frequencies from T. pallidum genome codon frequencies is described in Methods. Estimates of the translation rate benefit of codon bias for E. coli relative to low bias codon frequencies are shown by the black bars of Figure 1 , again assuming a short, invariant duration of translocation. The estimated benefits range from 0.2 – 0.6; as expected, these estimates are smaller than estimates derived from a comparison to strictly uniform codon usage. Because the theoretical estimates of Scenario 5 fall in the middle of the corresponding ranges of empirical estimates from Scenarios 1–4, we are confident that our results are not merely an artifact of unrecognized errors in the empirical rate measurements. The benefit of codon bias calculated for individual amino acids Our definition of s bias can be applied over any subset of codons, in particular, it can be applied to the codons of each amino acid separately. While all amino acids with multiple codons except proline contribute positively to s bias in all scenarios, the magnitude of that contribution is highly variable between amino acids (Figure 2 ). Codon bias accelerates the translation of most amino acids only slightly in E. coli , because most non-preferred codons are not particularly rare in the E. coli proteome, compared to the preferred synonym. For example, among the 9 amino acids encoded by 2 codons, on average the preferred codon is 2.9-fold more abundant than the non-preferred codon. Of these amino acids, asparagine shows the greatest difference between preferred and non-preferred codon frequencies, with GAC being 5.2-fold more abundant than GAU. Even if the disparity in codon-specific translation rates is unrealistically large, the ratio of the frequencies of preferred to non-preferred codons in E. coli constrains the maximum possible value of s bias . For asparagine, even if the preferred codon were translated instantaneously (i.e., infinitely faster than the non-preferred codon), the difference between using the non-preferred codon at 16% of asparagine residues in E. coli instead of at 50% of asparagine residues in Uni corresponds to only about a 3-fold acceleration of translation ( s bias ≈ 2) for this amino acid. With more realistic disparities between the translation rates of preferred and non-preferred codons, the largest estimate of s bias for asparagine in any of our scenarios is less than 0.2. In other words, we estimate that codon bias in E. coli leads to no more than a 20% decrease in the time required to translate all asparagine codons in the proteome (Figure 2 ). The amino acids with the largest values of s bias are leucine, isoleucine, and arginine (Figure 2 ). Although these amino acids are not rare, they possess between them the six rarest codons in E. coli , each encoding less than 0.1% of the proteome. (An average codon encodes 1.6% of the proteome.) The frequencies of the most and the least abundant synonyms for leucine, isoleucine and arginine differ by 74-fold, 83-fold, and 1460-fold, respectively. (The higher ratio for arginine reflects the extreme rarity of AGG, which is 17-fold less abundant than the second rarest E. coli codon, AUA encoding isoleucine.) Since the translation rates measured or assumed for the 6 rarest codons are quite slow, their increased abundance in Uni accounts for the much of the additional time required for replicating the Uni proteome. If these six codons remained as rare in Uni as they are in E. coli , while all other synonymous codons were used without bias in Uni, the translation rate benefit estimated under Scenario 4 (the scenario producing the largest estimate of s bias ) would be reduced by almost half (data not shown). The influence of these 6 codons is such that the estimate of s bias is quite sensitive to the translation rates assigned to them, in contrast to the relative insensitivity of s bias to the exact translation rates assigned to most codons. Discussion We want to know whether reduced codon bias could account for the lower translational power measured in at least some slowly growing bacteria, in comparison to E. coli . We approach this issue by its converse, calculating how much faster the proteome is replicated in E. coli than it would be in the complete absence of codon bias. If we take our estimates at face value, we would conclude that even during rapid growth when the proteome is most biased and translation is fastest, s bias is unlikely to be much larger than 1 (cross-hatched bars of Figure 1 ), which corresponds to a 2-fold increase in the average translation rate. An effect of this magnitude approaches the smaller disparities in the comparisons of translational power between E. coli and slowly growing strains shown in Table 1 , but could not explain the roughly 5-fold difference in translational power between E. coli and S. alaskensis , R. prowazekii , Halobacterium cutirubrum , or sulfate-reducing strain PT2. However, there are two reasons to think that the benefit of codon bias for E. coli , in comparison to most actual slow-growing organisms, is even less than this estimate. The first reason is that we have prevented our hypothetical Uni from adapting to the codon frequencies we have assigned to it, by keeping the abundance of each component of the translational apparatus fixed. The data do not suggest that maximizing translational power has been the only selective pressure influencing codon use in E. coli [ 45 , 65 ]. If it had been, the codon with the highest rate constant for ternary complex selection among synonymous alternatives would always be the preferred codon, since it would permit faster translation with a lower biomass investment in cognate tRNA. Of 10 amino acids with multiple codons for which codon-specific translation rate measurements exist [ 44 , 45 ], leucine, serine and proline are not consistent with this prediction. On the other hand, it seems clear that selection for rapid translation has exerted some, and perhaps the major influence on the coevolution of codon frequencies and tRNA abundance in E. coli . The codon with the highest rate constant is the preferred codon for 7 of the 10 amino acids for which data are available. Other considerations (possibly including error avoidance [ 66 ], interactions between adjacent tRNA anticodons [ 67 ], or factors unrelated to translation [ 68 ]) may have been more influential than the inherent characteristics of the codon-anticodon interactions for determining the preferred codons encoding leucine, serine and proline. However, the importance of rapid translation remains evident in that E. coli still translates the preferred codons quickly for 2 of these 3 amino acids, albeit with a larger investment in tRNA than would be necessary if the interaction between the preferred codon and its cognate tRNA occurred more readily. At a larger scale, the correlation across all codons between frequency and cognate tRNA abundance [ 69 , 70 ] is best explained as a response to selection for rapid translation, as is the pattern of increased bias towards rapidly translated codons with increased levels of gene expression [ 45 ]. Without asserting that the distribution of tRNA abundance in E. coli necessarily produces the fastest possible translation rate for the E. coli codon frequency distribution, it is clear that selection for translational power has been a significant factor in the co evolution of codon frequencies and cognate tRNA abundances in E. coli . Thus, it is very unlikely that we have attained the maximum possible translation rate for Uni by matching the E. coli distribution of tRNA abundance values (in the form of a particular distribution of codon-specific translation rates) to the very different codon frequency distribution of Uni. For this reason, our estimates confound the translation rate benefit of codon bias in E. coli with the penalty of a suboptimal allocation of translational resources in Uni. The second reason that our approach overstates the relative benefit of codon bias for E. coli in comparison to actual slow-growing organisms is that actual microbes are not completely devoid of codon bias. Assessing s bias in E. coli in comparison to a biologically plausible standard for low codon bias, instead of in comparison to the implausible standard of no codon bias whatsoever, reduces the estimated benefit in E. coli by about half (black bars of Figure 1 ). Only a slight bias in codon use is sufficient to obtain a substantial benefit of faster translation because only a few codons in E. coli are translated much more slowly than the median rate (Table 2 ). Moderate avoidance of only these few codons can provide a considerable acceleration of the average translation rate without generating a dramatic bias in overall codon use. Our estimate of a biologically plausible standard for low bias codon frequencies is deliberately conservative, underestimating the degree of bias expected in most slowly growing microbes, for two reasons. First, our low bias codon frequencies are based on the genome codon frequencies of T. pallidum , as if all predicted genes in the genome were expressed equally. Correspondence analysis performed at the level of individual genes failed to uncover evidence that codon use varies with expression level in T. pallidum [ 54 ]. If this were true, the proteome codon frequencies would indeed be similar to genome codon frequencies, regardless of variability in gene expression levels. However, a more sensitive analysis using codon frequencies summed over a set of putative high expression genes indicates that codon use in such genes is more biased than codon use in the genome as a whole. This conclusion is based on a comparison of Wright's effective number of codons [ 64 ] calculated for codon frequencies summed over all predicted genes annotated as ribosomal proteins or translation elongation factors (Nc = 52.7) or calculated for codon frequencies summed over all predicted genes in the genome (Nc = 55.2) [ 71 ]. The failure to observe this low level of codon bias in the previous analysis based on individual gene sequences [ 54 ] can probably be attributed to high gene-to-gene variability in codon frequency estimates based on the small samples of codons represented by individual genes. Thus, even for T. pallidum , the proteome codon frequencies appropriate for estimating the benefit of codon bias will be more biased than the genome-derived low bias codon frequencies shown in Table 2 . The second reason our low bias codon frequencies underestimate the degree of codon bias in most slowly growing microbes is that T. pallidum is essentially free of the influence of mutational bias, with a genome G+C content of 52.7%. In contrast, many slow-growing microbes have more extensive codon bias that can be attributed mostly or entirely to the biased nucleotide composition of the genome (e.g., R. prowazekii [ 52 ], H. pylori [ 55 ], Borrelia burgdorferi [ 54 ], Buchnera aphidicola [ 72 ], Mycoplasma genitalium [ 73 ], and Chlamydia species [ 74 ]). If codon bias derived from mutational bias, like codon bias derived from translational selection, permits more rapid translation for the same investment in translational machinery, the use of low bias codon frequencies derived from T. pallidum will underestimate the translation rate of many slow growing strains. We believe that codon bias derived from mutational bias does, indeed, have the potential to accelerate translation. The translation rate benefit of codon bias depends on matching preferred codons with cognate tRNAs that are abundant and/or that form 3 canonical base pairs [ 7 , 8 ]. Even when codon use is determined by mutational bias in the DNA replication and repair systems [ 46 ], not by selection acting simultaneously on codons and their cognate tRNAs via translation-associated effects, selection for translational power can influence the relative abundance and anticodon sequence of tRNA species. Relatively few mutations are sufficient to influence the identity and abundance of tRNA molecules in an organism, in comparison to the number of mutations required to influence proteome codon frequencies. (Consider that 45 mutations could allow a single mutation in the anticodon wobble position or in the regulatory region of many or even all tRNA genes, depending on the organism, while 45 mutations could alter the identity of less than 0.5% of the >9,000 codons in genes encoding ribosomal proteins and translational elongation factors.) Hence, the mutation-selection balance argument invoked to explain diminished codon bias in genes expressed at low levels in many strains [ 8 , 75 ] also suggests that the distribution of tRNAs can be influenced by translational selection that may be too weak to create a dramatic effect on codon usage. In fact, if codon use is biased in the same direction in all genes (as expected if the source of codon bias is mutational bias), instead of being biased only in highly expressed genes, it would increase the selective pressure for adaptation of the tRNA pool. Hence, it would be very surprising if the anticodons and the relative abundances of tRNA molecules in organisms with high or low G+C content did not reflect their biased use of codons. This prediction is confirmed by the only two studies we have found of tRNA abundance in microbes with extreme G+C content, involving Mycoplasma capricolum (25% G+C) [ 76 ] and Micrococcus luteus (74% G+C) [ 77 ]. M. capricolum , but not M. luteus , can be considered a constitutively slow-growing strain. As expected, cognate tRNA abundance in both organisms is correlated with codon frequency, both across all codons and within synonymous codon families [ 76 , 77 ]. For M. capricolum , this is accomplished largely without the tRNA gene dosage effects that are important for E. coli [ 70 ] and B. subtilis [ 78 ], since 28 of the 29 M. capricolum tRNA genes are present in only a single copy [ 76 ]. These examples indicate that selection for translational power is operative even for organisms in which the codon bias is determined by mutational bias instead of translational selection, and even for slowly growing organisms. Because codon bias from any source can be exploited to obtain higher translational power, the estimates of s bias for E. coli compared to codon frequencies derived from T. pallidum will overstate the benefit that exists for E. coli relative to most other slowly growing microbes that have greater mutational bias. In summary, we believe the translation rate benefit of codon bias in E. coli is likely to be less than 0.6 (see black bars of Figure 1 ) relative to an actual slow-growing organism that shows limited codon bias, such as T. pallidum , and substantially less than 0.6 relative to a slow-growing organism with more extensive codon bias. We do not mean to suggest that the advantage of translating as much as 60% faster than a competitor is unimportant. Clearly, the benefit of codon bias for E. coli must be substantial, considering that it arises from the aggregate effect of many thousands of preferred codons that are stably maintained in the E. coli genome, despite the randomizing influence of mutation acting at each individual codon. On the other hand, the influence of codon bias on the average translation rate is far smaller than the differences in translational power observed between microbes adapted to different ranges of growth rates. For differences in codon bias to explain the difference in translational power between E. coli and S. alaskensis , s bias would have to be about 5; to explain the difference between E. coli and R. prowazekii , s bias would have to be about 3. Is it possible that the comparisons of translational power presented in Table 1 are flawed? The colorimetric assays used for RNA and protein measurement in these studies are indeed dependent on procedural details, such that comparisons between laboratories and between studies are less reliable than comparisons within a study. Nonetheless, variation between species in the estimates of translational power presented in Table 1 do not appear to result simply from large random errors around a common mean. Estimates of translational power for slowly growing species with few rrn operons cluster around low values; the reverse is true for species capable of rapid growth with higher numbers of rrn operons. In addition, our own measurements of 10 bacterial species (including E. coli , S. alaskensis and 8 recent soil bacterial isolates) reproduce the same pattern; we have found differences in translational power that are comparable in magnitude to those shown in Table 1 [ 79 ]. Hence, we believe the comparisons in Table 1 are an adequate representation of the differences in translational power between rapidly growing and slowly growing microbes. Conclusions Because codon bias influences translational power, and because the degree of codon bias due to translational selection may differ systematically between rapidly growing and slowly growing strains, we investigated the parsimonious hypothesis that observed differences in translational power between microbial species could be explained by differences in the degree of codon bias. However, based on the analysis reported here, such an explanation is not plausible. Instead, differences in translational power between rapidly growing and slowly growing species suggest that the translational apparatus itself has different performance characteristics in rapidly growing and slowly growing microbes. Methods Translational power, translation rate and the active fraction of ribosomes Conceptually, we define translational power as the rate of protein synthesis in a cell or culture, normalized to the biomass invested in the protein synthesis system. We intend the term to be synonymous with 'translational efficiency' [ 4 , 5 , 8 ]; our rationale for departing from established terminology is provided in the Introduction. The protein synthesis system is comprised of ribosomes, elongation factors, tRNAs, tRNA synthetases, mRNAs, and numerous other components. Measuring the mass of the entire system is not trivial, because it includes a variable fraction of the cell's protein. However, since the protein synthesis system includes essentially all the cell's RNA, we follow Kjeldgaard and Kurland [ 1 ] in using RNA mass ( R ) as an index of the biomass invested in the entire system. For a culture in balanced, exponential growth, the instantaneous rate of increase of any culture component is d X /dt = μ X , where μ is the specific growth rate and X is the mass of the component present in the culture at that moment. Hence, μ P is the rate of protein synthesis in a culture containing mass P of protein. Thus, our quantitative measure of translational power is: This quantitative measure of translational power will be consistent with the conceptual definition as long as RNA is a nearly constant fraction of the mass of the entire protein synthesis system. Translational power reflects both the average translation rate and the fraction of active ribosomes in a cell or culture, which we demonstrate as follows, using the approach of chapter 6 of reference [ 34 ]. 'Translation rate' refers to the rate of amino acid polymerization of an active ribosome. The average translation rate of a cell or culture is the rate of amino acid polymerization in the entire culture divided by the total number of active ribosomes: We know that the mass rate of protein synthesis in a culture in balanced growth is μ P . Units of protein mass can be converted to a number of amino acids by dividing the protein mass by the average mass of an amino acid: number of amino acids polymerized per unit time = μ P /(average mass of amino acid) (3) The number of ribosomes in a culture containing a mass R of RNA can be found by multiplying R by the fraction of RNA that is ribosomal, and then dividing by the mass of RNA in a ribosome. However, only a fraction of these ribosomes are active at any given time. Thus: Substituting Equations (3) and (4) into Equation (2) yields: After rearranging terms in Equation (5), we have: where The quantity μ P/R in Equation (6) is the quantitative measure of translational power from Equation (1) [ 1 , 3 ]. From Equation (6), it is clear that translational power reflects both the average translation rate and the active fraction of ribosomes in a cell or culture. What of the term we have labeled C , implying a constant? The two quantities in the numerator, the mass of RNA in a ribosome and the average mass of an amino acid, are indeed constant or nearly constant, both within a strain at different growth rates, and across strains. However, despite the constant ribosomal fraction of RNA reported in reference [ 3 ], other data indicate that the rRNA fraction decreases from about 85% to about 75% as growth rate declines in E. coli from 1.7 hr -1 to 0.28 hr -1 [ 70 ], a result which is expected on theoretical grounds [ 4 , 65 ]. This variation is not dramatic; it would reduce translational power by only 12%, if the average translation rate and active fraction of ribosomes were unchanged. Data are also available from 2 of the 4 studies that have compared translational power between E. coli and a slowly growing strain. The rRNA fraction is reported as 84% for H. cutirubrum at specific growth rates of both 0.10 hr -1 and 0.05 hr -1 , after the authors made the deliberately generous assumption that messenger RNA comprises 5% of the total RNA [ 37 ]. The rRNA fraction is about 85% for R. prowazekii at a specific growth rate of ~0.07 hr -1 , after a correction is made for 2–3% messenger RNA [ 38 ]. These data suggest that variation between microbial species in the ribosomal fraction of RNA is limited, even when comparing species that grow at very different rates. Comparisons of translational power based on published data Table 1 summarizes comparisons of translational power between E. coli and all other bacterial and archaeal species for which we could find both the protein content and the RNA content of cultures growing at known rates. Throughout this work, E. coli is represented by the Bremer and Dennis data [ 3 ], which are typical of the data reported for E. coli in many other studies. Similarly, comparisons between E. coli and 2 closely related species of enteric bacteria, S. enterica and E. aerogenes , are made using only a single representative study for the latter strains, chosen from among several published reports. For the remaining species, only a single published study was available for comparison, except for one species represented by two studies, both of which are included. For strains not grown at 37°C, we assume that the growth rate, but not the macromolecular content, would be altered by growth in the same medium at a different temperature [ 80 ]. The growth rates reported for these strains were adjusted to the growth rates expected at 37°C using the linear range of the relationship reported in reference [ 56 ]. (Although this temperature-growth rate relationship was generated with E. coli , the comparison is mathematically identical whether the temperature correction is applied to E. coli or to the comparison strain.) The comparisons in Table 1 use the fastest growth rate for which data are available for the comparison organisms, and use data for E. coli growing at a rate such that it matches the comparison organism for investment in the translational apparatus. (For two of the comparison strains, translational power differed considerably between the fastest growth rates obtained in different culture conditions; both values are reported.) One of three measures was used to gauge the level of investment in the translational apparatus, depending on the quantity measured in the original study. The possible measures were the number of ribosomes per cell volume, the ratio of protein to ribosomal RNA, or the ratio of protein to total RNA. Values of these quantities for E. coli were interpolated between adjacent data points to estimate the growth rate at which E. coli made the same investment in the translational apparatus as the comparison organism. The translational power of the comparison organism at the fastest available growth rate was then expressed as a percentage of the translational power of E. coli at the 'same investment' growth rate. A comparison at similar investment levels reflects the expectation that the selective pressure to maximize translational power increases with the biomass invested in the apparatus [ 4 , 58 ]. If the comparisons had always been made to the fastest E. coli growth rate (i.e., where E. coli translational power is highest), the disparities in translational power would be greater for most of the comparisons shown. Calculation of the translation rate benefit of codon bias Consider a cell in which a total of C i codons of type i are translated during a single cell generation, so that the sum over all sense codons C = Σ C i is the total number of codons translated during a cell generation. (Hereafter we refer to the translational output over a cell generation as the proteome.) If we define c i = C i / C as the proportion of all codons of type i in the proteome and r i as the average translation rate of codons of type i , the total time required for replication of the proteome (i.e., the proteome generation time) will be where R # is the average number of ribosomes active in translation over the cell cycle and the sum is over all sense codons. Codon bias in favor of rapidly translated codons will reduce g p in comparison to uniform codon use. If a mutation changes the fitness of an organism from w to w ', the benefit of the mutation is typically described as s , where w '/ w = 1 + s . By analogy, and considering g p to be inversely related to fitness, we can express the translation rate benefit of codon bias as The protein content (and thus C ) is the same in Uni as in E. coli by hypothesis. With the restrictive condition that the abundance of each individual component of the translational apparatus is unchanged in Uni, ribosome content ( R # ) will be the same also. Hence, the C / R # term of g p in Equation (8) cancels from both the numerator and denominator of Equation (9) for s bias , leading to Since amino acid frequencies are identical in E. coli and Uni, the disparities in translation rates between synonymous codons largely determine the magnitude of the translation rate benefit of codon bias. We will use the same codon-specific translation rates (the r i 's) for both Uni and E. coli , again invoking the restrictive stipulation that the abundance of each individual tRNA species is unchanged. If rate constants for the interaction of each codon with each of its cognate tRNA species were known, we could calculate the optimal tRNA abundance distribution for the codon frequencies of Uni, and infer the resulting codon-specific translation rates [ 62 , 65 ]. However, in vivo codon-specific translation rate data are available only as codon averages, including translation from all tRNA species cognate to each codon. Hence, rate constants specific to each codon-cognate tRNA pair cannot be calculated from the available data for the codons translated by multiple tRNA species, and thus we cannot calculate an optimal tRNA abundance distribution for Uni. Instead, we have constrained Uni to maintain the same tRNA distribution and codon-specific translation rates as E. coli . Insofar as the E. coli rates reflect an allocation of tRNA abundance that would be sub-optimal for Uni (as we argue in the Discussion section), our approach will tend to overestimate of the benefit of codon bias in E. coli , a conservative error for our purposes. Data sources All data used in our estimates of s bias are reported in Table 2 . For the codon frequencies used in synthesizing the proteome of E. coli , we rely on the data of Dong et al . at a specific growth rate of 1.73 hr -1 [ 70 ], compiled from public gene sequence databases and protein abundance data derived from 2D gel electrophoresis studies [ 81 , 82 ]. The absolute codon frequencies shown in Table 2 have been recalculated from [ 70 ] with initiation and stop (including selenocysteine) codons removed. As expected, the translation rate benefit of codon bias was found to increase monotonically with growth rate, when calculated by any of the scenarios described below, using the proteome codon frequencies and tRNA abundance data from the range of growth rates reported in reference [ 70 ] (data not shown). This increase in s bias reflects simply the increasing bias in both proteome codon usage and relative tRNA abundance with increasing growth rate. Since we are interested in the maximum effect of codon bias, we report results from only the highest growth rate for which data are available. To investigate the importance of low levels of codon bias, we applied Equation (10) either with Uni having strictly uniform use of synonymous codons, or with Uni assigned a set of low bias codon frequencies (Table 2 ). The low bias frequencies were generated from relative codon frequencies over all predicted genes in the complete genome sequence of T. pallidum [ 71 ]. By relative codon frequencies, we mean the absolute frequency of a codon divided by absolute frequency of the amino acid it encodes. The set of T. pallidum relative codon frequencies for a particular amino acid were multiplied by the absolute frequency of that amino acid in the E. coli proteome; the resulting set of absolute codon frequency values were assigned to the codons of that amino acid in the low bias set so as to retain the same rank order of codon frequency among synonyms as exists in the E. coli proteome. For example, the absolute frequency of isoleucine and the identity of the 1st, 2nd and 3rd most common isoleucine codons are the same in the low bias set as in the E. coli proteome. However, the relative frequencies of the 1st, 2nd and 3rd most common isoleucine codons in the low bias set are the same as the relative frequencies of the 1st, 2nd and 3rd most common isoleucine codons in the T. pallidum genome. To represent codon-specific translation rates, we use the relative rate data (the quantity R tRNA /R shift ) of Curran and Yarus [ 45 ] for the 29 sense codons beginning with U or C (YNN codons, Y = pyrimidine). Although incomplete, this is by far the largest data set available for in vivo translational kinetics. The original publication transposed values reported for two arginine codons, CGC and CGA [ 83 ]; we have corrected this error. We also revised the rate measured for CGA downward, to account for interference from the bulky wobble position inosine-adenine base pair in the P site that results from translation of a CGA codon. Such interference is strongly suggested to slow selection of a ternary complex at the codon subsequent to CGA [ 83 ]; such an effect would not have been measured with the experimental system of reference [ 45 ], but is appropriate to include as a codon-specific effect of CGA on translation rate. In the absence of more precise data, we reduced the translation rate measured for CGA by a factor of 3, the factor by which CGA reduces read-through of a following stop codon by a suppressor tRNA in comparison to CGC [ 83 ]. This adjustment to the CGA rate brings these results into rough agreement with those of Sorensen and Pedersen [ 84 ], who used an experimental approach that would have detected a consistent effect of CGA on the translation rate of the subsequent codon, attributing it to slow translation of CGA itself. The relative rates of reference [ 45 ], modified as described above, are listed in Table 2 . The relative rates reported by Curran and Yarus [ 45 ] do not reflect the entire translational cycle, but rather the time required for selecting a cognate ternary complex at an empty, codon-programmed ribosomal A site, which is believed to occupy the majority of the elongational cycle [ 63 ]. Although peptide bond formation may be very rapid, the time required for the EF-G-catalyzed translocation of the ribosome to the subsequent codon (and the associated movement of P- and A-site tRNAs) may not be much shorter than the time needed for EF-Tu-catalyzed ternary complex selection [ 63 ]. Hence, in addition to calculations made using rates of ternary complex selection to represent an entire cycle of translational elongation (assuming, in effect, that the duration of translocation is negligible), we also made calculations after modifying the reported rates by adding an invariant 'translocation time' to the variable 'ternary complex selection time' for all codons. The duration of translocation per codon was set at 40% of the average time required to select a ternary complex containing tRNA phe at a UUU codon, consistent with the only quantitative measure of translocation rate that has been made in conditions approximating those in vivo [ 63 ]. Results from both sets of calculations (white and cross-hatched bars of Figure 1 ) are presented for each scenario (described below) that is based on these ternary complex selection rates. For convenience, elsewhere in this report we refer to the relative rates of reference [ 45 ] as translation rates, rather than using the more accurate but cumbersome expression 'ternary complex selection rates'. To calculate the total abundance of cognate tRNA for each codon, we assign cognate specificity largely according to Björk [ 85 ], and use the tRNA abundance data from Dong et al . [ 70 ]. We differ from Björk only in assuming that the leucine and glycine tRNAs with uridine in the anticodon wobble position (for which nucleotide modifications have not been characterized) will read codons ending in U, A and G, instead of A and G only. This would be the case if the wobble position U is modified to cmO 5 U, as is done for each of the other 6 amino acids encoded by a full box of the translational code (i.e., amino acids for which the four XXN codons are synonyms). Following Björk, we assume that 40% of the tRNAs for glutamate, glutamine and lysine with uridine in the anticodon wobble position are modified to mnm 5 Se 2 U and thus read codons ending in A or G; the balance of these tRNA species are assumed to have mnm 5 S 2 U in the wobble position and read A-ending codons only [ 85 ]. The abundance of two pairs of isoaccepting tRNA species (Gln1 + Gln2 and Ile1 + Ile2) were reported as summed values by Dong et al . [ 70 ], since these individual species were not separated under the experimental conditions applied. We have resolved the summed values to the abundance of individual species using the ratios of the individual abundance values reported by Ikemura [ 69 ]. We show cognate tRNA abundance data in Table 2 as a percentage of total tRNA, omitting initiator and selenocysteine tRNAs; the sum of all values is greater than 100%, reflecting the partially overlapping specificity of many tRNA species. Scenarios for extrapolating from incomplete empirical translation rate data We address the incompleteness of codon-specific translation rate data in several ways. In Scenario 1, we assume that the effects of biased use of YNN codons on translation rate can be used to represent the effects of bias over all codons, without assigning particular translation rates to the unmeasured codons. However, since the YNN codons are almost half of all sense codons but only account for about a third of all expression (Table 2 ), they must be less highly expressed, on average, than the RNN codons (R = purine). Consequently, selection for translational power may have been weaker among YNN codons than RNN codons. Scenarios 2–4 address this potential deficiency by applying various strategies of assigning translation rates to the unmeasured codons that are consistent with observed patterns, but that could allow the effect of codon bias on translation rate to be greater among RNN codon than YNN codons. Scenario 5 abandons empirical codon-specific translation rate measurements completely, assigning translation rates to all codons on the basis of the proteome codon frequency and cognate tRNA abundance of E. coli , assuming optimality (i.e., maximal translation rate) according to theory developed by Solomovici et al . [ 62 ]. Scenario 1 The 29 YNN codons encode 10 amino acids, 9 of which have multiple codons. For 7 of these 9 amino acids, the most common synonym is the codon with the fastest translation rate. One of the remaining amino acids is serine, for which the two fastest-translated codons are the two most abundant, although in reverse order, with relatively small differences between the two in both rate and abundance. Only proline appears to be anomalous; the 2 most abundant codons encode over 90% of all proline residues in the proteome [ 70 ], but support ternary complex selection about 3.5-fold more slowly than the 2 least abundant codons [ 45 ]. It has been suggested [ 45 ] that this anomaly could be adaptive; if proline, because of its unique structure, is found preferentially between protein domains [ 86 ] where slow translation may be important to permit cotranslational folding [ 87 , 88 ]. If proline is the only amino acid for which such contrarian selection pressure is more important than selection for translational power, including proline codons in a sample intended to represent all codons will lead to an underestimate of s bias . Hence, in Scenario 1 we apply Equation (10) over YNN codons, with the calculated translation time for non-proline YNN codons weighted by a factor of 3.2, which scales the expression level of these codons to the expression level of all non-proline codons. In other words, we assume the effects of codon bias on translation rate among the 25 non-proline YNN sense codons are representative of the effects of codon bias among all 57 non-proline sense codons, whereas the translation rates measured for proline codons are applied only to themselves. Scenario 2 Curran and Yarus noted that among highly expressed genes, there is a significant tendency for rapidly-translated codons to be used frequently, although the relationship appears to be nonlinear [ 45 ]. We observe the same pattern comparing their relative rate data to the proteome codon frequency data of Dong et al . [ 70 ] at the highest growth rate. For non-proline YNN codons, the best fit (R 2 = 0.56) of a quadratic relationship passing through the origin between the codon frequency and translation rate data of Table 2 is c i = 0.205 r i - 0.522 r i 2 . We use this equation to predict translation rates from codon frequency for all RNN codons, as shown in Table 2 . Since our objective is to obtain a reasonable estimate the codon-specific translation rate for codons which have not been measured, not to defend a particular model of the relationship between codon frequency and translation rate, we make no attempt to justify a quadratic fit in comparison to other possible functional relationships. The predicted rates for RNN codons and the measured rates for YNN codons (Table 2 ) are used with Equation (10) to estimate the translation rate benefit of codon bias under Scenario 2. Scenario 3 The preceding scenario applied to the YNN codons tends to predict translation rates among synonymous alternatives that are not as disparate as those actually observed. Furthermore, the fit of a functional relationship between codon frequency and translation rate among YNN codons is better when only preferred codons are considered, instead of all codons. Hence, we fit a quadratic relationship passing through the origin to data from 10 preferred non-proline YNN codons, obtaining c i = 0.352 r i - 1.611 r i 2 (R 2 = 0.81). Among the 10 preferred codons, we include UGG, the sole tryptophan codon, and UUG, the preferred leucine codon within the UUR split box although not the preferred leucine codon overall. We then apply this equation to predict translation rates from codon frequencies for 12 preferred RNN codons, including AUG, the sole methionine codon, and AGG and AGC, the preferred arginine and serine codons within their respective split boxes, although not the preferred codons overall. For non-preferred RNN codons, translation rate is predicted by multiplying the predicted rate for the preferred synonym (within the full or split box) by the ratio of the square roots of the codon frequencies for the non-preferred and preferred codons: This relationship was chosen both because a dependence on the square root of codon frequency has been suggested repeatedly in theoretical investigations of optimal translation rates [ 62 , 65 , 89 , 90 ], and because for all non-preferred RNN codons, this relationship leads to a greater disparity of predicted translation rates compared to the preferred synonym than the regression of Scenario 2. (It also predicts a greater translation rate disparity than is observed for the majority of non-preferred YNN codons.) When both the quadratic regression for preferred codons and Equation (11) for non-preferred codons are applied to predict the translation rate of non-proline YNN codons, the correlation of predicted with measured translation rates is comparable to that attained with Scenario 2 (R 2 = 0.57). The predicted rates for RNN codons and the measured rates for YNN codons (Table 2 ) are used with Equation (10) to estimate the translation rate benefit of codon bias under Scenario 3. Scenario 4 This scenario is generated in three steps, with the goal of generating an estimate of the translation rate benefit of codon bias that is consistent with the most extreme empirical observations. First, three rare RNN codons (AGG and AGA for arginine and AUA for isoleucine, all with c i < 0.1%) are assigned the slowest relative translation rate observed among YNN codons ( r i = 0.6 for the rare leucine codon CUA). Second, the translation rates for preferred RNN codons within full or split boxes (except AGG) are estimated according to the regression equation described for Scenario 3. Finally, the translation rates for non-preferred codons (except AGA and AUA) are predicted from the preferred synonym using the ratios of the most disparate translation rates observed empirically among synonymous alternatives, treating split boxes and full boxes of the translational code separately. The most extreme ratio observed among translation rates in a split box is 3.375, for glutamate codons in the study of Sorensen and Pedersen [ 84 ]. The most extreme ratios observed for translation rates of codons in a full box is 1:1.3:1.6:24 for the CUN leucine codons in the study of Curran and Yarus [ 45 ]. (Exploring other rate values 1 ≤ x ≤ y ≤ 24 in ratios of the form 1: x : y :24 failed to find any that greatly increased the estimated benefit beyond that using the leucine ratios, data not shown.) Although this scenario is based on extreme observations, applying these 3 rules to the non-proline YNN codons leads to a correlation of predicted and measured translation rates (R 2 = 0.67) somewhat better than that obtained under Scenario 2 or Scenario 3. The predicted rates for RNN codons and the measured rates for YNN codons (Table 2 ) are used with Equation (10) to estimate the translation rate benefit of codon bias under Scenario 4. Scenario 5 In contrast to the preceding scenarios that extend codon-specific translation rate measurements of YNN codons in various ways to make estimates of the effect of codon bias over all codons, Scenario 5 incorporates a theoretical prediction of the optimal translation rates for all codons based only on codon frequency and cognate tRNA abundance data. While this approach necessarily involves additional assumptions, it has the advantage of drawing on data that is more complete and less likely to be influenced by unrecognized experimental errors. Solomovici et al . [ 62 ] assume that selection on synonymous codon frequencies reflects intrinsic differences in rate constants for a cognate tRNA interacting with preferred and non-preferred codons, while the total tRNA abundance and amino acid composition are fixed. They demonstrate that the fastest overall translation rate is obtained when the square roots of synonymous codon frequencies are proportional to the rate constants for cognate tRNA interacting with the codons. They assume further that the rate constants for the interaction of all non-degenerate or preferred codons with their preferred cognate tRNA are identical, so the translation rate for these codons is proportional to cognate tRNA abundance. We modified the approach of reference [ 62 ] to reflect greater degeneracy in translation than assumed by the original authors ([ 85 ], also the comments earlier in this section), and applied it using the codon frequency and tRNA abundance data of Dong et al . [ 70 ], modified as shown in Table 2 . The predicted relative translation rates for YNN codons (i.e., the recalculated quantities d ij and d im, j of reference [ 62 ] for codons with single or multiple cognate tRNAs, respectively) are not in good agreement with observed relative rates of Curran and Yarus [ 45 ] (R 2 = 0.30). However, the empirical codon frequencies of Dong et al . [ 70 ] are correlated more closely with predicted relative rates of Scenario 5 (R 2 = 0.70) than with the empirical relative rates of Curran and Yarus [ 45 ] (R 2 = 0.31). A good correlation between the predicted translation rates and the empirical codon frequencies is expected, since the codon frequencies were used to generate the predictions. However, the poor correlation between predicted and empirical translation rates could reflect the inadequacies in any of 3 areas: 1) the assumptions of Solomovici et al . [ 62 ], 2) the rate measurements of Curran and Yarus [ 45 ], and/or 3) the codon and tRNA data of Dong et al . [ 70 ]. Alternatively, the discrepancy between predicted optimal translation rates and empirical rates may indicate that the phenotype of E. coli is not perfectly optimized for maximal translation rates (as suggested in reference [ 65 ]), either because of genetic drift or because of conflicting selection pressures. Nonetheless, the disparity between the relative rates of synonymous preferred and non-preferred codons for most amino acids are greater with the predicted rates of Scenario 5 than with the observed rates. Hence, Scenario 5 will generate a higher estimate of the translation rate benefit of codon bias than would a strict application of the empirical codon-specific translation rates. (In fact, none of our scenarios are strict applications of the empirical rates; Scenarios 1–4 also deliberately extrapolate from the empirical rates in ways that will increase the estimated benefit of codon bias.) The predicted translation rates for all codons (Table 2 ) are used with Equation (10) to estimate the translation rate benefit of codon bias under Scenario 5. Authors' contributions LD conceived of the project, collected and analyzed the data, developed the mathematical model, and drafted the manuscript. TMS helped plan the project, critiqued the work as it progressed, and edited the manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546186.xml |
314464 | Cell-Passage Activity Is Required for the Malarial Parasite to Cross the Liver Sinusoidal Cell Layer | Liver infection is an obligatory step in malarial transmission, but it remains unclear how the sporozoites gain access to the hepatocytes, which are separated from the circulatory system by the liver sinusoidal cell layer. We found that a novel microneme protein, named sporozoite microneme protein essential for cell traversal (SPECT), is produced by the liver-infective sporozoite of the rodent malaria parasite, Plasmodium berghei . Targeted disruption of the spect gene greatly reduced sporozoite infectivity to the liver. In vitro cell invasion assays revealed that these disruptants can infect hepatocytes normally but completely lack their cell passage ability. Their apparent liver infectivity was, however, restored by depletion of Kupffer cells, hepatic macrophages included in the sinusoidal cell layer. These results show that malarial sporozoites access hepatocytes through the liver sinusoidal cell layer by cell traversal motility mediated by SPECT and strongly suggest that Kupffer cells are main routes for this passage. Our findings may open the way for novel malaria transmission-blocking strategies that target molecules involved in sporozoite migration to the hepatocyte. | Introduction Malaria is one of the most devastating infectious diseases in the world, killing more than 1 million people per year. Malaria is transmitted by bites of infected mosquitoes that inject sporozoites under the skin. The first obligatory step for these parasites to establish infection in humans is migration to hepatocytes, where they proliferate and develop into the erythrocyte-invasive form ( Sinnis 1996 ). This liver-invasive stage has been demonstrated as a promising target for antimalarial strategies that aim to establish sterile immunity against the malarial parasite ( Nussenzweig et al. 1967 ; Hoffman et al. 1996 ). However, the mechanisms underlying the parasite's liver infection are largely unknown. In particular, it has been controversial how sporozoites reach the hepatocytes that are separated from blood circulation by the liver sinusoidal layer. The routes the sporozoites use to cross this layer, the modes of motility on which their migration is based, and the molecules of the parasite involved in this process are poorly understood. Malarial parasites develop into sporozoites in the mosquito midgut and then invade the salivary gland, where they wait to be transferred to the mammalian host ( Menard 2000 ). Once injected by mosquito bites under the skin, sporozoites enter the blood circulation and are carried to the liver by the bloodstream ( Sinnis and Nussenzweig 1996 ; Menard 2000 ; Mota and Rodriguez 2002 ). In the liver, they are thought to be arrested on the inner surface of the liver sinusoidal vein and then leave the vein and infect the hepatocytes by crossing the sinusoidal wall ( Sinnis and Nussenzweig 1996 ). This wall is a single-cell layer mainly composed of sinusoidal endothelial cells and Kupffer cells, which are hepatic macrophages. Several models have been proposed to explain how sporozoites cross this layer. Some authors proposed that sporozoites infect hepatocytes after crossing the liver endothelial cell through fenestrations in this cell ( Vanderberg and Stewart 1990 ), but these openings are too small for sporozoites to freely pass through ( Mota and Rodriguez 2002 ). Other authors have suggested that Kupffer cells are gates for sporozoites to access hepatocytes, based on the ultrastructural observation that sporozoites were found inside Kupffer cells shortly after intravenous inoculation ( Mota and Rodriguez 2002 ). This Kupffer cell hypothesis, however, has not been convincingly demonstrated, because other tools for probing into this event were lacking. Furthermore, the observation that the sporozoites in Kupffer cells sometimes have a vacuole around them makes the conclusion uncertain. Some authors have proposed that sporozoites are passively engulfed by Kupffer cells and then carried to the hepatocyte ( Meis et al. 1983 ), and some have proposed that this migration is based on active motility accompanied by vacuole formation ( Pradel and Frevert 2001 ). The malarial parasite has no locomotory organelles such as flagella or cilia. Motility of the host-invasive stages of the malarial parasite, including the sporozoite, is dependent on secretion of micronemes that are organelles occupying the cytoplasm of the parasite ( Sultan 1999 ; Menard 2001 ). Micronemal components, which may include several attachment proteins, are secreted from the apical pore during parasite movement and are translocated backwards along the parasite cell surface by actomyosin motors of the parasite. This surface movement is believed to generate traction for parasite-invasive motility. Salivary gland sporozoites display three modes of motility in vitro dependent on secretion of micronemes ( Mota and Rodriguez 2002 ). One mode is gliding motility on a solid surface, which can be observed as circular movement on a glass slide, probably representing gliding motility on the cell surface. The other two are cell-invasive motilities: cell-infection and cell-traversal motility ( Mota et al. 2001 ; Kappe et al. 2003 ). Cell-infection motility is accompanied by vacuole formation and is followed by parasite development into exoerythrocytic forms (EEFs). Cell-traversal motility, on the other hand, involves plasma-membrane disruption and is followed by migration through the cytoplasm and eventual escape from the cell. Recently, Mota et al. (2002 ) revealed that this type of cell-invasion motility can be identified by conventional cell-wound assay. According to the observation that passage through some hepatocytes by this motility precedes hepatocyte infection, they proposed the hypothesis that this motility is necessary for sporozoites to be activated for hepatocyte infection ( Mota et al. 2002 ). However, the role of this motility in liver infection remains unclear. Aiming at identification of molecules involved in sporozoite infection, we screened an expressed sequence tag (EST) database of the salivary gland sporozoite of a rodent malarial parasite, Plasmodium berghei . In this paper, we report a novel microneme protein, named SPECT (sporozoite microneme protein essential for cell traversal), which is specifically produced by the liver-infective sporozoite and is essential for the sporozoite's cell-passage ability. By using spect -disrupted parasites, we show that cell-passage ability of the sporozoite plays a critical role in malarial transmission to the vertebrate host and is required for sporozoites to access hepatocytes by traversal of the liver sinusoidal cell layer. In addition, we provide a model of sporozoite liver infection, which suggests an answer to the question of how sporozoites reach the hepatocytes. Results Identification of cDNA Encoding SPECT from P. berghei Salivary Gland Sporozoite EST Database Sporozoites acquire the ability to infect the mammalian liver after infection of the mosquito salivary glands ( Sultan et al. 1997 ), indicating that novel protein synthesis for liver infection begins in this stage ( Matuschewski et al. 2002 ). To search for malarial genes involved in liver infection, we screened an EST database of P. berghei salivary gland sporozoites. We assembled 3,825 ESTs, obtained 502 contigs, and screened them for genes encoding secretory proteins or membrane-associated proteins, which may participate in host–parasite interactions. This screening was started from the contigs containing a high number of ESTs, since the number of ESTs may correlate with the expression levels of the respective genes. In this process, we identified a contig composed of ten ESTs, encoding a putative secretory protein of 241 amino acids ( Figure 1 A). The expected molecular mass for the N-terminal signal sequence-processed form of this protein was 25 kDa. We named this protein SPECT (sporozoite microneme protein essential for cell traversal), since it is essential for sporozoite passage through a host cell, as described later. Figure 1 Sequence Analysis of spect cDNA (A) Nucleotide sequence of spect cDNA (top lane) and the deduced amino acid sequence (bottom lane) are shown. The predicted N-terminal signal sequence is underlined. The numbers indicate positions of the nucleotides starting from the 5′ end. The asterisks indicate the termination codon. (B) A comparison of deduced amino acid sequences of P. berghei spect (top) and P. falciparum spect (bottom). Gaps are introduced to obtain optical matching by using GENETIX-MAC software. Asterisks or dots show conserved or similar residues, respectively. The amino acid numbers from the first Met residue are shown on the left of each line. Southern blot analysis showed that the spect gene is a single-copy gene (data not shown). Sequence analysis of the spect gene identified four introns (data not shown). A computer search of Plasmodium genome databases ( Carlton et al. 2002 ; Gardner et al. 2002 ) revealed that this gene is conserved through several Plasmodium species. The orthologous protein in P. falciparum, the clinically most important human malaria parasite, shared 45.6% sequence identity with P. berghei SPECT ( Figure 1 B). SPECT Is Produced Specifically by Salivary Gland Sporozoites and Localized in Micronemes The expression profile of this gene in the malarial life cycle was investigated. Immunofluorescent analysis in all host-invasive stages showed that SPECT production was restricted to sporozoites in the salivary gland ( Figure 2 A). It is noteworthy that SPECT is not detected in sporozoites in the midgut, because this expression profile strongly suggests that SPECT is specifically involved in liver infection. Western blot analysis revealed SPECT as a 22 kDa protein in salivary gland sporozoites, but not in midgut sporozoites ( Figure 2 B), confirming that SPECT is produced after invasion into the salivary gland. Immunoelectron microscopy showed that SPECT is localized in the sporozoite to micronemes that are secretory organelles occupying the cytoplasm ( Figure 2 C). Micronemes are common to motile stages of Plasmodium parasites and play a central role in host-invasive motility ( Sultan 1999 ; Menard 2001 ). Taken together, these results indicate that SPECT plays a role in the liver-invasive motility of the sporozoite. Figure 2 SPECT Is a Microneme Protein Specifically Produced in the Liver-Infective Sporozoite Stage (A) Indirect immunofluorescence microscopy of all four invasive forms of the malarial parasite (indicated over the panel). Parasites were stained with primary antibodies against SPECT, followed by FITC-conjugated secondary antibodies. SPECT was detected only in the salivary gland sporozoite, the liver-infective stage. The corresponding phase-contrast or DAPI-stained image (Phase or DAPI) is shown under each image. Scale bars, 5 μm (B) Western blot analysis of SPECT production in the midgut sporozoite (M) and the salivary gland sporozoite (S). Lysate of 500,000 sporozoites was loaded onto each lane and detected with the same antibody used in (A). SPECT was detected as a single band of 22 kDa (arrowhead) only in the salivary gland sporozoite. (C) Immunoelectron microscopy of sporozoites in the salivary gland. Ultrathin sections of a mosquito salivary gland infected with sporozoites were incubated with the same antibody used in (A) followed by secondary antibodies conjugated with gold particles (15 nm). Particles were localized to micronemes (Mn) but not to rhoptories (Rh). Axial (inset) and vertical images are shown. Scale bars, 0.5 μm. SPECT Plays an Important Role in Sporozoite Infection of the Host Liver To investigate the function of SPECT protein, we generated spect -disrupted parasites by homologous recombination ( Figure 3 A). The spect disruptants were selected by the antimalarial drug pyrimethamine and were separated from wild-type parasites by limiting dilution. Disruption of the spect locus was confirmed by Southern blot analysis ( Figure 3 B). To exclude the possibility that the spect -disrupted populations obtained were derived from a single clone, two independently obtained spect -disrupted populations ( spect (−)1 and spect (−)2) were used in the following experiments. Figure 3 Targeted Disruption of the spect Gene (A) Schematic representation of targeted disruption of the spect gene. The targeting vector (top) containing a selectable marker gene is integrated into the spect gene locus (middle) by double crossover. This recombination event resulted in the disruption of the spect gene and confers pyrimethamine resistance to disruptants (bottom). (B) Genomic Southern blot hybridization of wild-type (WT) and spect (−) populations. Genomic DNA isolated from the respective parasite populations was digested with EcoT22I and hybridized with the probe indicated in (A) by a solid bar. By integration of the targeting construct, the size of detected fragments was decreased from 1.9 kbp to 1.2 kbp. The result is shown for two independently prepared populations, spect (−)1 and spect (−)2. (C) Immunofluorescence microscopy of the wild-type (WT) and spect (−) parasite. Sporozoites were collected from the salivary gland and stained with primary antibody against SPECT followed by FITC-conjugated secondary antibodies. The apical end of each sporozoite is indicated by an arrowhead. In the intra-erythrocytic stage, SPECT gene disruption did not affect parasite proliferation, as the growth rates in rat blood were almost the same in the spect -disrupted and wild-type populations (data not shown). Furthermore, disruption of the gene did not affect parasite development in the mosquito vector, as numbers of sporozoites residing in the midgut and in the salivary glands were similar in the spect -disrupted and wild-type populations ( Table 1 ). Table 1 SPECT Disrupted Parasites Develop Normally into Sporozoites and Invade the Salivary Gland in the Mosquito Vector Mosquitoes were fed on mice infected with spect (−) parasite populations or wild-type polyclonal populations. Sporozoites were collected separately from the midgut and the salivary glands of mosquitoes 24–28 d after feeding and then counted. Each value is the mean of the number with its standard error from three independent experiments Next, the liver infectivity of the spect -disrupted sporozoites was examined. Rats were intravenously inoculated with sporozoites, and the progress of parasitemia, the percentage of infected erythrocytes, was measured in the exponential growth period (from 3.5 d to 5 d after inoculation). It is thought that the parasitemias reflect the liver infectivity of the respective parasite populations, since the growth rates of their intraerythrocytic stages are similar (shown by the parallel slopes of the increase in parasitemia in Figure 4 ). Based on the average parasitemia at 3.5 d after inoculation of 30,000 sporozoites, the liver infectivities of the two disruptant strains were estimated to be 15- and 28-fold lower, respectively, than that of the wild-type. These results are consistent with the observation that the parasitemias after injection of 30,000 disruptant sporozoites were lower than that from 3,000 wild-type sporozoites. Figure 4 Targeted Disruption of spect Results in Reduction of Sporozoite Infectivity to the Liver (A) The salivary gland sporozoites of each parasite population were injected intravenously into five rats. The parasitemia of each rat was checked by a Giemsa-stained blood smear after inoculation on the days indicated. The average parasitemia after inoculation of 30,000 sporozoites was significantly low in disruptant populations, whereas their growth rates in the blood were essentially the same as the wild-type. The numbers of parasites inoculated were as follows: 30,000 spect (−)1 (open circles), 30,000 spect (−)2 (open triangles), 30,000 wild-type (filled circles), and 3,000 wild-type (filled squares). Values shown represent the mean parasitemia (± SEM) of five rats. (B) The salivary gland sporozoites (500,000) of wild-type or spect -disrupted parasites were inoculated intravenously into 3-wk-old rats. After 24 h, the livers were fixed with paraformaldehyde and frozen. The number of EEFs on each cryostat sections was estimated by indirect immunofluorescence analysis using anti-CS antiserum. Values shown represent the mean number of EEFs per square millimeter (± SEM) of at least three rats. The liver infectivity was also evaluated by the number of early EEFs. Frozen sections of the rat liver was prepared 24 h after sporozoite injection and EEFs were counted by immunofluorescence microscopy. As shown in Figure 4 B, EEFs were approximately 30-fold decreased by spect gene disruption. This reduction rate agrees well with that estimated by parasitemia. These results indicate that SPECT plays a role in the process of sporozoite invasion into the liver. SPECT Is Essential for Sporozoite Cell-Passage Ability Localization of SPECT in micronemes indicates its involvement in the invasive motility of the sporozoite. The motility of spect -disrupted sporozoites was investigated by three in vitro assays corresponding to three modes of motility of the sporozoite. First, we checked gliding motility on a solid surface, which is essential for sporozoite infectivity. Most disruptants displayed a typical circular movement, and the proportion of motile sporozoites was almost identical in disruptant and wild-type parasites (63.6% and 67.5%, respectively), showing that their gliding motility is not affected by SPECT gene disruption. Second, we examined the ability of the sporozoites to infect hepatocytes. This was assayed by formation of EEFs in a human hepatoma cell line, HepG2 ( Hollingdale et al. 1981 ). As shown in Figure 5 A, the disruptants formed EEFs in similar numbers to the wild-type, indicating that they retain normal infectivity to the hepatocyte. Third, we examined cell-traversal ability that takes place prior to hepatocyte infection. This was estimated by the number of membrane-wounded cultured cells that were labeled by uptake of fluorescein isothiocyanate (FITC)-conjugated dextran from the medium ( Mota et al. 2001 ). As shown in Figure 5 B, the cell-wound assay using HeLa cells showed that the disruptants lost their cell-passage activity completely. The same results were obtained in HepG2 cells (data not shown). These results revealed that SPECT is specifically involved in cell-traversal ability and suggest that lack of this ability reduced liver infectivity of the disruptants. Figure 5 spect Disruption Results in Loss of Cell-Passage Activity of the Sporozoite (A) spect disruption does not affect sporozoite ability to infect hepatocytes. (Top panel) Comparison of EEF numbers between disruptants ( spect (−)) and wild-type (WT) parasites. Salivary gland sporozoites were added to HepG2 cells and cultured for 48 h. EEFs formed were counted after immunofluorescence staining with an antiserum against CS protein. (Bottom panels) Representative fluorescence stained images. (B) Sporozoites lacking SPECT cannot traverse HeLa cells. (Top) Comparison of cell-passage activity between disruptants and wild-type parasites. Salivary gland sporozoites were added to HeLa cells and incubated for 1 h with FITC-conjugated dextran (1 mg/ml). Cell-passage activity was estimated by the number of cells wounded by sporozoite passage, which were identified by cytosolic labeling with FITC-conjugated dextran. (Bottom) Representative fluorescence stained images. All data are mean numbers of EEFs or FITC-positive cells in a one-fifth area of an 8-well chamber slide with standard errors for at least three independent experiments. Cell Passage Ability Is Necessary for Sporozoites to Traverse the Sinusoidal Layer Cells and to Access Hepatocytes To access the hepatocytes, sporozoites must cross the sinusoidal layer, which separates them from the circulation. We assumed that SPECT was necessary for this process. Since Kupffer cells are major components of this layer and have been reported as the main gates for sporozoite access to the hepatocyte, we prepared Kupffer cell-depleted rats by intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl 2 MDP) ( Vreden et al. 1993 ; van Rooijen and Sanders 1994 ) and tested them for infection by disruptant and wild-type sporozoites. As shown in Figure 6 A, infectivities of spect -disruptants assessed by parasitemia were increased by 22- and 53-fold by Kupffer cell depletion and, as a result, became equal to that of the wild-type. The numbers of early EEFs detected in the liver sections were also almost identical in wild-type and spect -disrupted parasites ( Figure 6 B). These results show that the disruptants' loss of infectivity is localized at the sinusoidal cell layer and that the cell-passage ability of the sporozoite is necessary to cross this layer and, specifically, the Kupffer cells. Figure 6 Restoration of spect (−) Sporozoite Infectivity in Kupffer Cell-Depleted Rats (A) Liposome-encapsulated Cl 2 MDP (filled points) or PBS (open) was injected intravenously into rats. After 48 h, 30,000 sporozoites of spect (−)1 (circles), spect (−)2 (triangles), or wild-type (squares) populations were inoculated intravenously. Parasitemia of each rat was checked by Giemsa-stained blood smears after inoculation on the days indicated. Values shown represent the mean parasitemia (± SEM) of five rats. (B) Salivary gland sporozoites (500,000) of each parasite population were inoculated intravenously into Kupffer cell-depleted rats. After 24 h, the livers were fixed with paraformaldehyde and frozen. The number of EEFs on each cryostat section was estimated by indirect immunofluorescence analysis using anti-CS antiserum. Values shown represent the mean number of EEFs per square millimeter (± SEM) of at least three rats. Discussion It has been reported that the Plasmodium sporozoite has the ability to traverse cultured cells rapidly ( Mota et al. 2001 ), but the role of this process in liver infection has remained unclear. On the other hand, it is poorly understood how the sporozoite migrates from the circulatory system to the hepatocyte. In this paper, we address these issues using a gene-targeting technique. We have shown that the cell-traversal activity of the sporozoite is necessary for it to leave the circulatory system by crossing the liver sinusoidal cell layer. These results are the first to reveal the role of cell-traversal activity in malarial transmission. In vitro cell invasion assays showed that spect -disrupted sporozoites completely lose cell passage activity, but preserve normal infectivity to the hepatocyte (see Figure 5 ). These results clearly demonstrated that these two cell-invasion activities are independent of each other. This conclusion contradicts the hypothesis proposed by Mota et al. (2002 ) that cell passage activates the sporozoite for hepatocyte infection. They assumed that sporozoites traverse some hepatocytes before infecting a hepatocyte and that this passage alters their mode of cell invasion from passage to infection ( Mota et al. 2002 ). Our results, however, demonstrated that lack of previous cell passage has no influence on the infectivity to hepatocytes. This independence was confirmed in vivo by the result that disruptants and wild-type showed the same liver infectivities in Kupffer cell-depleted rats (see Figure 6 ). Therefore, sporozoites may change their mode of invasive motility according to other factors, which remain to be elucidated. We suppose that secretion of the micronemal contents during gliding on the cell surface might be one such factor, since this motility may precede hepatocyte infection as discussed below. Our results indicate that the liver sinusoidal barrier is not perfect, since a small proportion of the spect -disrupted sporozoites can infect the liver (see Figure 4 ). It is supposed that this layer may have a few openings and the disruptants can migrate through them by gliding along the epithelial cell surface. In Kupffer cell-depleted rats, on the other hand, both disruptants and wild-type may migrate through the numerous gaps created among the endothelial cells, resulting in elimination of the phenotypic difference. Since Kupffer cells constitute approximately 30% of the sinusoidal cells ( Bouwens et al. 1986 ), their depletion from this layer may leave many gaps that cannot readily be repaired. Supposedly, sporozoites cross these gaps in the same way as they migrate through the few gaps in normal rats. Experiments using Kupffer cell-depleted rats indicate that Kupffer cells are not involved in sporozoites targeting the liver, because the depletion did not reduce the susceptibility of rats to sporozoite infection. Thus, sporozoites seem to be first arrested on the endothelial cell surface or on the glycosaminoglycans extending through endothelial fenestrations and then migrate to Kupffer cells ( Cerami et al. 1992 ; Pradel et al. 2002 ). If so, gliding motility on the cell surface would be necessary for the sporozoite to migrate from initial attachment sites to Kupffer cells (or to gaps) along the inner surface of the sinusoidal layer as well as for the sporozoite to migrate through gaps. These assumptions imply that after Kupffer cell depletion, sporozoites can arrive at the hepatocyte by gliding motility alone, in accord with the observation that the disruptants can infect Kupffer cell-depleted rats with the same infectivity as the wild-type. Our results strongly suggest that Kupffer cells are main gates for sporozoites to access hepatocytes. Previous electron microscopic studies have reported that sporozoites are observed in Kupffer cells after intravenous inoculation, and some of them are found within vacuoles ( Meis et al. 1983 ; Pradel and Frevert 2001 ). Based on this observation, it has been speculated that sporozoites invade the Kupffer cell by a motility distinct from passage that does not involve parasitophorous vacuole formation. Our results, on the contrary, indicate that sporozoites cross the layer by the same cell-passage motility as observed in vitro. We think this discrepancy indicates the following two possibilities. One is that the vacuole formed in the Kupffer cell after rupture of its cell membrane is different from the parasitophorous vacuole formed in the hepatocyte, although their differences cannot be distinguished by electron microscopy. Another possibility is that the parasites seen in vacuoles were phagocytosed ones and not in the process of invasion. In fact, if their invasion mode is cell-traversal motility, as we believe, this event may be rapidly completed and difficult to catch by electron microscopy. Therefore, many phagocytosed parasites could be included among those seen. Taking the evidence together, we propose that the sporozoites access the hepatocyte through Kupffer cells by the same cell-traversal motility that has been identified in vitro, and we propose a model for sporozoite liver infection in Figure 7 . Figure 7 Schematic Representation of Sporozoite Migration to and Infection of Hepatocytes (Left) Sporozoites migrate to the space of Disse through the Kupffer cells. [1] The sporozoite (Sp) in the circulatory system is sequestered to the sinusoidal endothelial cell (EC) by specific recognition of the cell surface or glycosaminoglycans extending from the hepatocytes (He) through fenestration. [2] The sporozoite begins to glide on the epithelial cell surface. [3] Encountering a Kupffer cell (KC), the sporozoite ruptures the plasma membrane, passes through the cell, and enters into the space of Disse. Thus, the sporozoite gains access to hepatocytes. This step requires SPECT. [4] The sporozoite infects a hepatocyte with formation of a vacuole and develops into EEF in the hepatocyte. (Right) An alternative route to the hepatocyte. A small number of sporozoites, which find gaps in the sinusoidal layer while gliding, migrate to hepatocytes directly through the openings without need for cell passage and infect the hepatocytes. Likewise, in Kupffer cell-depleted rats, both wild-type and spect (−) sporozoites can enter hepatocytes through numerous gaps present between the sinusoidal endothelial cells. In this study we have established the significance of cell-passage ability of the sporozoite in malaria transmission and have demonstrated that this ability is necessary for breaking through the liver sinusoidal barrier. Cell-traversal activity plays an important role in other invasive stages of the malarial parasite, including the ookinete, which migrates through the epithelial cells of the mosquito midgut, and the sporozoite in the oocyst, which is released from the mature oocyst and then migrates through the salivary gland cell. Our study revealed that another cellular barrier is present in the malarial life cycle and sporozoites must break through this barrier by cell-traversal activity. Our recent work has identified two other genes that are involved in the cell passage activity of the sporozoite. Like SPECT, the products of these genes have a secretory protein-like structure and are localized in the micronemes. Furthermore, sporozoites disrupted for these genes have similar phenotypic character to spect -disrupted ones, including impaired cell-passage ability, decreased liver infectivity with similar reduction rate, complete restoration of the infectivity in Kupffer cell-depleted rats, normal gliding motility, and normal hepatocyte infectivity (unpublished data). This suggests that the cell-traversal ability of the sporozoite is realized by cooperation of several microneme proteins. We suggest that these molecules could be targets for antimalarial strategies, since success in crossing this layer is critical for the malarial parasite to establish infection in humans. Elucidation of the molecular mechanisms of passage may lead to novel malaria transmission-blocking strategies that prevent sporozoites from gaining access to the hepatocyte. Materials and Methods Parasite preparations Female 6–10-wk-old BALB/c mice (Japan SLC, Inc., Hamamatsu, Japan) infected with the P. berghei ANKA strain were prepared by peritoneal injection of infected blood that was stored at −70°C. For the purification of sporozoites, infected mosquitoes were dissected 24–28 d after the infective blood meal. The salivary glands and midgut were separately collected in medium 199 on ice and then gently ground to release the sporozoites. Ookinetes and erythrocytic-stage parasites were prepared as described previously ( Yuda et al. 1999 ; Kariu et al. 2002 ). Genomic Southern blot hybridization Genomic DNA of P. berghei parasites (2 μg) was digested with ClaI, EcoRI, EcoT22I, HindIII, or XbaI, separated on 1.2% agarose gel and then transferred to a nylon membrane. DNA fragments were amplified by PCR using genomic DNA as template with the following primers: 5′-TGGGCAATTTTGCCTTTAAAAACG-3′ and 5′-TTTCATTGTGTTAAACGATAAGTG-3′. They were labeled with [ 32 P]dCTP and used as probes. Antibody preparation and Western blot analysis Recombinant SPECT without signal sequence was expressed as a glutathione S-transferase (GST)–fusion protein using the pGEX 6p-1 system (Amersham Bioscience, Uppsala, Sweden). The recombinant protein was purified with a GST column and used for immunization of rabbits. Specific antibodies were affinity purified using a N-hydroxysuccinimide-activated column (Amersham Bioscience) coupled with recombinant SPECT protein. For CS antiserum production, the peptide DPPPPNANDPAPPNAN, corresponding to the repeat region, was conjugated to keyhole limpet hemocyanin as a carrier and used for the immunization of rabbits. Western blot analysis was performed as described previously ( Kariu et al. 2002 ). Immunofluorescence microscopy and immunoelectron microscopy Immunofluorescence microscopy was performed as described previously ( Kariu et al. 2002 ). Purified parasites were fixed in acetone for 2 min. The slides were incubated with anti-SPECT rabbit antibodies and then with FITC-conjugated secondary antibody (Zymed. South San Francisco, California, United States). For nuclear staining, 4′,6-diamidino-2-phenylindole (DAPI) (0.02 μg/ml final concentration) was added to the secondary antibody solution. Immunoelectron microscopy was performed as described previously ( Yuda et al. 2001 ). In brief, purified parasites were fixed in 1% paraformaldehyde–0.1% glutaraldehyde for 15 min on ice. After embedding in LR Gold resin (London Resin Company Ltd., London, United Kingdom), ultrathin sections were incubated with anti-SPECT antibodies and then with secondary antibody conjugated to gold particles (15 nm diameter) (AuroProbe, Amersham Pharmacia Biotech, Uppsala, Sweden). The samples were examined with a Hitachi H-800 transmission electron microscope (Hitachi, Tokyo, Japan) at an acceleration voltage of 100 kV. Targeted disruption of the spect gene For construction of the targeting vector, two fragments of the spect gene were amplified by PCR using genomic DNA as template with the primer pairs 5′-CGCGAGCTCGCAATATGGTATTAAATTTTGGGCTAGCCA-3′ and 5′-CGCGGATCCGGTATTTTCATTGTGTTAAACGATATGTGA-3′ and 5′-CCGCTCGAGGTCCTATTTATCATTTTAAAATGTGTTTTATC-3′ and 5′-CGGGGTACCAATCGTCATAAATAGGAGTTATGAAGT-3′. These fragments were cloned into either side of the selectable marker gene in pBluescript (Strategene, La Jolla, California, United States). The gene targeting experiment was performed as described previously ( Yuda et al. 1999 ). Evaluation of sporozoite infectivity to rats Sporozoites collected from mosquito salivary glands were suspended in medium 199 and then injected intravenously into 3-wk-old female Wistar rats (Japan SLC, Inc., Hamamatsu, Japan) ( n = 5). Before each inoculation, sporozoites were checked for their ability to glide in vitro to confirm that they contained over 60% motile sporozoites. Parasitemia was checked every 12 h by a Giemsa-stained blood smear. Measurement of the number of EEFs in the infected liver Sporozoites (5.0 × 10 5 ) were intravenously inoculated into a 3-wk-old female Wistar rat. After 24 h, the liver was perfused with PBS followed by 4% paraformaldehyde. The liver was further fixed in 4% paraformaldehyde for 6 h and frozen in liquid nitrogen. Cryostat sections (20 μm) were prepared from the left lobe and fixed in acetone for 2 min on a glass slide. The EEFs were detected by immunofluorescence staining using rabbit anti-CS antiserum and FITC-conjugated secondary antibody. At least 12 sections were examined under an Olympus (Tokyo, Japan) BX60 fluorescence microscope (200×) and the number of EEFs per square millimeter was calculated. EEF development assay in vitro The EEF formation assay was performed as described previously ( Hollingdale et al. 1981 ) with minor modifications. HepG2 cells (5.0 × 10 5 ) were plated in 8-well chamber slides. Sporozoites (5.0 × 10 3 or 5.0 × 10 4 ) were suspended in 100 μl of complete medium and added to this culture. After 2 h, the media were replaced with 400 μl of fresh complete medium supplemented with 3 μg/ml glucose. The slides were incubated for 2 d with medium changed twice a day and were fixed in acetone for 2 min. The EEFs were detected by immunofluorescence staining as described above. The number of EEFs in one-fifth of the area of each well was counted under an Olympus BX60 fluorescence microscope (200×). Cell-traversing activity assay The traversing activity of the sporozoite was examined using a standard cell-wounding and membrane repair assay ( Mota et al. 2001 ). HepG2 cells (2.5 × 10 5 ) or HeLa cells (5.0 × 10 4 ) were inoculated into 8-well chamber slides (Nunc Inc., Napierville, Illinois, United States). Sporozoites were added 2 d later to cells for 1 h in the presence of 1 mg/ml FITC-labeled dextran (10,000 MW, lysine-fixable; Molecular Probes, Inc., Eugene, Oregon, United States). The cells were incubated for an additional 3 h in complete culture medium and fixed with 4% paraformaldehyde in PBS. The number of FITC-positive cells was counted under a fluorescence microscope. Depletion of rat Kupffer cells For depletion of Kupffer cells, 3-wk-old female Wistar rats were injected intravenously with 120 μl of liposome-encapsulated Cl 2 MDP or an equal volume of PBS as control. After 48 h, sporozoites were injected into a tail vein and the parasitemia was checked by Giemsa-stained blood smears. Cl 2 MDP liposomes were prepared as described elsewhere ( van Rooijen and Sanders 1994 ). Elimination of Kupffer cells was confirmed by immunoperoxidase staining after liver perfusion with PBS followed by fixation with 4% paraformaldehyde in PBS. Cl 2 MDP was a gift from Roche Diagnostics (Mannheim, Germany). | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC314464.xml |
521690 | The pattern of methacholine responsiveness in mice is dependent on antigen challenge dose | Background Considerable variation exists in the protocols used to induce hyperresponsiveness in murine models of allergic sensitisation. We examined the effect of varying the number of antigen exposures at challenge on the development of methacholine responsiveness in systemically sensitised mice. Methods BALB/c mice were sensitised with ovalbumin (OVA), challenged with 1, 3 or 6 OVA aerosols. Lung function was measured using low frequency forced oscillations and partitioned into components representing the airways (R aw ) and lung parenchyma (tissue damping (G) and tissue elastance (H)). Responsiveness to inhaled methacholine (MCh), inflammatory cell profile and circulating IgE were assessed 24 and 48 hours after challenge. The threshold dose of MCh required to elicit a detectable response (sensitivity) and response to 30 mg.mL -1 (maximal response) were determined for each compartment. Results Sensitivity ; All three OVA protocols resulted in an increased sensitivity to MCh in R aw but not in G or H. These responses where present at 24 and 48 hrs, except 1 OVA aerosol in which changes had resolved by 48 hrs. Maximal response ; 1 OVA aerosol increased maximal responses in R aw , G and H at 24 hrs, which was gone by 48 hrs. Three OVA aerosols increased responses in H at 48 hrs only. Six OVA challenges caused increases in R aw , G and H at both 24 and 48 hrs. Eosinophils increased with increasing antigen challenges. IgE was elevated by OVA sensitisation but not boosted by OVA aerosol challenge. Conclusions The pattern of eosinophilia, IgE and MCh responsiveness in mice was determined by antigen dose at challenge. In this study, increased sensitivity to MCh was confined to the airways whereas increases in maximal responses occurred in both the airway and parenchymal compartments. The presence of eosinophilia and IgE did not always coincide with increased responsiveness to inhaled MCh. These findings require further systematic study to determine whether different mechanisms underlie airway and parenchymal hyperresponsiveness post antigen challenge. | Background Persistent asthma is an allergic disease characterised by airway inflammation ([ 1 - 5 ]) and hyperresponsiveness to external stimuli ([ 1 ]). Mouse models of allergic airway sensitisation are often used to elucidate the pathobiology of this disease ([ 6 - 8 ]). To date, a number of techniques have been used to measure changes in lung function in response to bronchoconstricting agents in murine models of allergic bronchopulmonary inflammation (see [ 6 , 8 , 9 ] for reviews). One method that has gained recent popularity is unrestrained barometric plethysmography, which uses a 'pseudo-flow' measurement to derive a dimensionless parameter known as enhanced pause (Penh). There are now several publications in the literature which claim to have documented airway hyperresponsiveness in allergen-driven murine models based on methacholine induced changes in Penh. However, it has also been well documented that Penh does not correlate with changes in the physiology of the lung ([ 10 - 14 ]), especially in chronic disease states ([ 15 ]). In contrast, the low frequency forced oscillation technique (LFOT) is able to provide sensitive measurements of respiratory system input impedance (Zrs) in the mouse, that are partitioned into components representing airway and parenchymal compartments by fitting the constant-phase model ([ 16 - 18 ]). Using LFOT, Tomioka et al . ([ 17 ]) found that systemic sensitisation followed by three antigen challenges, one of the most common allergen models utilised in studies using Penh, resulted in hyperresponsiveness that was confined primarily to the tissue compartment of the lung. This has important implications for the interpretation of results obtained with Penh that have demonstrated mechanisms underlying allergic inflammation in mice given that a significant portion of the respiratory system hyperreactivity to MCh in human asthmatics is a result of the response of the conducting airways ([ 19 ]). One of the most common methods for inducing allergic bronchopulmonary inflammation in mice involves systemic sensitisation with a specific antigen and Th-2 skewing adjuvant, usually ovalbumin (OVA) adsorbed onto aluminium hydroxide (Alum), followed by airway challenge with the same antigen ([ 20 - 22 ]). However, considerable variations exist between studies in terms of the dose of antigen used during airway challenge. To date, a number of studies have found that airway hyperresponsiveness is increased by increasing the dose of antigen at challenge ([ 23 - 25 ]). However, these studies, which used different doses of antigen at challenge as part of a broader intervention protocol, have used Penh ([ 23 , 24 ]) or a measure of total lung resistance ([ 25 ]) to examine the resulting changes in lung physiology. As yet, no studies have systematically examined the effect of the dose of antigen at challenge on the subsequent development of hyperresponsiveness using a technique that is able to partition the reactivity of the lungs into airways and tissue compartments. Hyperresponsiveness of the respiratory system to bronchoconstricting agents, and other outcome parameters such as those that reflect inflammation and allergic sensitisation, are often measured at different times post challenge. In an examination of the kinetics of hyperresponsiveness in an OVA model of allergic sensitisation in mice using a single dose of antigen at challenge, Tomkinson et al . ([ 26 ]) found that responsiveness to methacholine (MCh) is maximal 24 hours post challenge, has begun to resolve by 48 hours, and has returned to baseline levels beyond that time. The kinetics of responsiveness to MCh in other studies, however, are often overlooked and it is yet be determined if altering the dose of antigen at challenge has an influence on the timing of peak responsiveness to bronchoconstricting agents. The aim of this study was to systemically investigate the effect of antigen dose at challenge on the pattern of hyperresponsiveness to inhaled MCh in a murine model of allergic bronchopulmonary inflammation. Methods Animals 8 week old specific pathogen free female BALB/c mice were purchased from the Animal Resources Centre, Murdoch, Western Australia. Mice were housed in a controlled environment with a 12 hr light:dark cycle and provided with an OVA free diet and acidified water ad libitum . All experiments were approved by the Institutional Animal Ethics and Experimentation Committee. Sensitisation protocols Mice were sensitised by intraperitoneal (i.p.) injection with 20 μg of OVA (Sigma, St Louis, USA) suspended in 200 μL of Alum (Alu-gel-S, Serva, Heidelberg, Germany) on days 0 and 14. Mice were then challenged with either 1, 3 or 6 OVA (1% w/v in PBS) aerosols delivered with an ultrasonic nebuliser (UltraNeb ® , DeVilbiss, Somerset, Pennsylvania) for 30 minutes on consecutive days starting at day 21 (Fig 1 ). Two additional groups of mice served as controls; a naïve group and a group sensitised with i.p. OVA and challenged with a single PBS aerosol using the protocol described above. Figure 1 Timeline for sensitisation and data collection. Timeline for the protocols used to induce allergic bronchopulmonary inflammation and timing for bronchoalveolar lavage (BAL), serum IgE measurement and assessment of hyperresponsiveness to inhaled methacholine (MCh). Mice were systemically sensitised with two intraperitoneal injections of OVA/Alum on day 0 and 14, challenged with either 1 (A), 3 (B) or 6 (C) OVA aerosols (1%) for 30 minutes starting at day 21. Respiratory mechanics Changes in Zrs were measured using a modification of the low frequency forced-oscillation technique (LFOT) as described previously ([ 27 ]). Briefly, mice were anaesthetised with an i.p. injection of a solution containing xylazine (2 mg.mL -1 , Troy Laboratories, NSW, Australia) and ketamine (40 mg.mL -1 , Troy Laboratories, NSW, Australia) at a dose of 0.01 mL.g -1 . Mice were tracheostomised with a 10 mm section of polyethylene tubing (1.27 mm OD: 0.86 mm ID) and ventilated ( flexiVent , Scireq, Montreal, Canada) at 450 b.min -1 with a tidal volume of 8 mL.kg -1 and a positive end expiratory pressure (PEEP) of 2 cmH 2 O. The lung volume history of the mice was standardised prior to measurement of lung mechanics using two deep inflations and three P-V curves. The respiratory system input impedance (Zrs) was measured during periods of apnea using a 16 s signal containing 19 mutually prime sinusoidal frequencies ranging from 0.25 to 19.625 Hz. The constant phase model ([ 16 ]) was then fit to the real and imaginary parts of the Zrs spectrum allowing the calculation of airway resistance (R aw ), tissue damping (G), tissue elastance (H) and hysteresivity (η) ([ 28 ]). Methacholine responsiveness Changes in respiratory mechanics following inhaled MCh were measured either 24 or 48 hrs after the last OVA aerosol. Following measurement of baseline Zrs, mice were exposed to a 90 s saline aerosol delivered with an ultrasonic nebuliser (UltraNeb ® , Devilbiss, Somerset, Pennsylvania). Zrs was then measured every minute for the next 5 minutes. This aerosol procedure was repeated with 1/2 log 10 incremental doses of MCh from 0.1 to 30 mg.mL -1 with Zrs measured every minute for at least 5 minutes after the aerosol until the parameters calculated from the constant phase model had peaked. Inflammatory cell counts Separate groups of mice, sensitised using the same protocol described above, were anaesthetised and tracheostomised 24 or 48 hrs after their last aerosol. BAL fluid was collected by slowly infusing and withdrawing a 1 mL aliquot of PBS containing BSA (bovine serum albumin, 20 mg.mL -1 , CSL, Victoria, Australia) and lidocaine (35 mg.mL -1 , Sigma, St Louis, USA) from the lungs three times. The BAL was then centrifuged at 2000 rpm for 4 mins. The supernatant was removed and the pellet resuspended in PBS. The cells were stained with trypan blue to determine viability and the total cell count (TCC) obtained by counting the cells with a haemocytometer. Differential counts were obtained from the cytospin sample, stained with Leishman's stain and examined using light microscopy. Three hundred cells were counted from each sample to determine the relative proportions of each cell type. Serum IgE In a separate group of mice, serum samples were periodically collected for analysis of total IgE. An additional control group was included in the analysis of serum IgE consisting of mice sensitised with PBS/Alum. Sera were diluted 1:7.5 in Delfia Assay buffer (Wallac Oy, Turku, Finland). The diluted sera were analysed for the presence of total IgE by time-resolved fluorescence (TRF) assays. Briefly, 96-well plates (Nunc Maxisorp, Denmark) were coated overnight at 4° C with anti-mouse IgE (R35-72; BD PharMingen, San Diego, USA). Plates were blocked with 200 μl of 0.5% BSA in TRIS-HCl pH 7.4 for 1 hour at room temperature on a plate shaker. For all subsequent steps a volume of 50 μl per well was used and incubations were performed for 1 hour at room temperature unless otherwise indicated. Between steps, plates were washed five times with wash buffer (TRIS-HCl pH 7.8 Tween20). Mouse anti-TNP IgE (BD PharMingen, San Diego, USA) was used as an interassay standard. Biotinylated anti-mouse IgE (R35-118; BD PharMingen, San Diego, USA) was added to the wells at 2 μg.mL -1 . Straptavidin-conjugated Europium (Wallac Oy, Turku, Finland) was incubated at 1:500 for 30 minutes and plates washed eight times thereafter. Delfia enhancement solution (Wallac Oy, Turku, Finland) was added and the plates were agitated on a shaker for 10 minutes prior to reading the fluorescence on a Wallac Victor 2 counter (Wallac Oy, Turku, Finland). The detection limit of this assay is approximately 100 ng.mL -1 . Statistical analysis Log 10 transformed inflammatory cell and immunoglobulin data were compared using ANOVA and Tukey's post-hoc test. Responses in R aw and G to inhaled MCh at the maximum dose used (30 mg.mL -1 ) were expressed as a percentage of the response to the saline aerosol and compared using non-parametric ANOVA on ranks and Dunn's post-hoc test. Responses in H were expressed as a percentage of the response to saline, log 10 transformed and compared using ANOVA and Tukey's post-hoc test. The threshold dose of MCh where there was a detectable change in R aw , G or H (termed sensitivity hereafter) was interpolated from the raw dose response curve as the upper limit of the 99% CI of the 5 measurements taken following the saline aerosol (Fig. 2 ). The sensitivity data were compared using ANOVA and Tukey's post-hoc test. All data were analysed using SigmaStat 2.03 and p values < 0.05 were deemed to be significant. Figure 2 Technique for sensitivity calculation. Schematic representation of the technique used for calculation of the threshold dose of MCh (sensitivity) required to induce a detectable increase in R aw , G and H. Results Methacholine responsiveness The degree and time of observed maximum MCh induced responses in R aw , G and H varied substantially between treatments (Fig. 3 ). A summary of statistical comparisons of sensitivity to MCh and percentage response to the maximum dose (30 mg.mL -1 ) between treatment groups and naïve mice is presented in Table 1 . Sensitisation followed by challenge with a single PBS aerosol did not cause an increase in sensitivity or maximum responsiveness to MCh compared to naïve mice. Figure 3 Dose response curves to inhaled methacholine. Dose response curves (expressed as a % of the response to saline aerosol) for mice systemically sensitised with OVA/Alum and challenged via the airways with 1 (left), 3 (centre) or 6 (right) OVA aerosols. Mice were challenged 24 (●) or 48 (▲) hours after the last OVA aerosol. Dose response curves from naïve mice (○) are also shown. All data are expressed as mean ± SEM (n = 7–8). * indicates significance (p < 0.05 vs naïve mice; ANOVA on Ranks, Dunn's post-hoc for R aw and G; ANOVA, Tukey's post-hoc for H). Table 1 Summary of sensitivity and maximum responses to methacholine in airway and parenchymal lung compartments. Summary of the threshold dose (sensitivity) required to elicit a detectable increase in airway resistance (R aw ), tissue damping (G) and tissue elastance (H) for naïve mice, mice systemically sensitised with OVA/Alum and challenged with PBS and mice systemically sensitised and challenged with OVA. Also shown is the percentage change in R aw , G and H in response to the maximum does of methacholine used (30 mg.mL -1 ). Data are presented as the mean (SEM). Challenge Assessed after last aerosol (hr) Sensitivity - Threshold dose of MCh (mg.mL -1 ) Response at 30 mg.mL -1 MCh Raw G H Raw § G § H § p* p* p* p* p* p* Naïve - 0.54(0.14) - 0.51(0.25) - 0.10(0.02) - 235.5(21.5) - 138.8(6.1) - 145.4(5.1) - 1 PBS aerosol 24 and 48 pooled 0.55(0.30) ns 0.25(0.10) ns 0.06(0.01) ns 242.9(19.1) ns 141.5(4.7) ns 143.4(2.7) ns 1 OVA aerosol 24 0.09(0.03) 0.012 0.15(0.04) ns 0.05(0.01) ns 514.0(82.7) <0.05 275.5(30.6) <0.001 250.2(24.5) <0.001 48 0.46(0.19) ns 0.18(0.05) ns 0.17(0.07) ns 326.4(32.6) ns 213.0(14.6) ns 179.3(12.6) ns 3 OVA aerosols 24 0.12(0.03) 0.012 0.35(0.20) ns 0.06(0.01) ns 271.2(43.1) ns 180.3(41.7) ns 178.9(39.9) ns 48 0.20(0.07) 0.034 0.16(0.04) ns 0.08(0.01) ns 348.8(46.0) ns 243.6(42.7) ns 233.0(31.6) 0.045 6 OVA aerosols 24 0.17(0.05) 0.019 0.12(0.05) ns 0.06(0.01) ns 456.7(43.4) <0.05 358.2(88.4) <0.05 298.5(52.2) 0.02 48 0.16(0.05) 0.034 0.15(0.06) ns 0.06(0.01) ns 396.0(23.3) <0.05 304.8(28.5) <0.05 295.6(30.6) 0.018 § expressed as a % of saline response * vs naïve values One OVA aerosol A single OVA aerosol was sufficient to induce a significant increase in MCh responsiveness in the airways, seen as both a lower threshold dose of MCh required to induce a response (increased sensitivity) and increased response at the 24 hour time point (Table 1 ). In the parenchymal compartment, no increase in sensitivity was seen but a significant increase in maximal response was seen for both G and H. This heightened sensitivity and response had diminished, back to the level seen in naive mice, 48 hours after the OVA aerosol. Three OVA aerosols Three OVA aerosols resulted in significantly increased airway (but not parenchymal) sensitivity to MCh at both the 24 and 48 hour time points (Table 1 ). However, there was no increase in maximum response at 24 hours in R aw , G or H and only an increased response in H after 48 hours but not R aw and G. Six OVA aerosols Six OVA aerosols resulted in both significantly increased airway sensitivity and maximal responses to MCh at 24 and 48 hours post-challenge. Increased maximal responses, but not increased sensitivity, were also seen in the parenchymal compartment at both the 24 and 48 hour time points. Inflammatory cell counts Challenge with a single PBS aerosol following systemic sensitisation with OVA did not cause a significant increase in TCC in the BAL (p = 0.552) compared to naïve mice (Fig. 4 ). There was, however, a significant increase in TCC in mice challenged with a single OVA aerosol (p = 0.032) and a further increase in TCC following 3 OVA challenges (p < 0.001). Exposure to 6 OVA aerosols did not cause any further increase in TCC above levels observed in mice exposed to 3 OVA aerosols (p = 0.805) but remained significantly higher than mice challenged with 1 OVA aerosol (p < 0.001). Time of sampling after the last aerosol with any of the protocols did not have a significant impact on TCC (p = 0.357). Figure 4 Total cell counts from bronchoalveolar lavage. Total cell counts (TCC) from the bronchoalveolar lavage (BAL) of naïve BALB/c mice, mice systemically sensitised and challenge with OVA aerosols and mice systemically sensitised with OVA and challenged with PBS. Samples were collected 24 (grey) and 48 (black) hours after the last aerosol. Data are expressed as mean ± SEM (n = 5–6). Exposure to a PBS aerosol following antigen sensitisation did not cause an increase in TCC (p = 0.552). In contrast, a single OVA aerosol was sufficient to cause a significant increase in TCC (p = 0.032). Exposure to 3 OVA aerosols caused a further increase in TCC (p < 0.001) but 6 OVA aerosols did not cause an increase in TCC beyond those observed in mice exposed to 3 OVA aerosols (p = 0.805). The number of aeroallergen challenges also had a significant impact on the number of eosinophils (p < 0.001) and macrophages (p < 0.001) in the BAL. There were significant increases in the number of eosinophils in sensitised mice challenged with 1 (p = 0.032), 3 (p < 0.001) and 6 (p < 0.001) OVA aerosols (Fig. 5 ) compared to naïve mice. The numbers of eosinophils in the BAL of mice exposed to 3 and 6 aerosols were significantly higher than those exposed to a single OVA aerosol (p < 0.001 and p < 0.001 respectively) but were not significantly different from each other (p = 0.805). The number of macrophages in the BAL were also higher in mice exposed to 3 (p < 0.001) and 6 (p < 0.001) OVA aerosols compared to naïve mice. As with TCC, time of sampling after the last aerosol did not have a significant impact on the number of eosinophils (p = 0.357) or macrophages (p = 0.079) in the BAL. Low levels of neutrophils were observed in BALs from OVA challenged mice sampled at 24 hours but not in mice sampled 48 hours after the last OVA aerosol (Fig. 5 ). Lymphocyte numbers were not significantly elevated in the BALs from any of the treatment groups ( data not shown ). Figure 5 Differential cell counts from bronchoalveolar lavage. Differential cell counts from the bronchoalveolar lavage (BAL) of naïve BALB/c mice, mice systemically sensitised and challenge with 1,3 or 6 OVA aerosols and mice systemically sensitized with OVA and challenged with a single PBS aerosol. BALs were collected 24 and 48 hours after the last aerosol. Data are expressed as mean ± SEM (n = 5–6). There was a significant increase in the number of eosinophils (p = 0.032) in the BAL following a single OVA aerosol. Exposure to 3 or more OVA aerosols caused a further increase in the number eosinophils (p < 0.001), compared to 1 OVA aerosol, and an increase in the number of macrophages (p < 0.001) compared to naïve mice. There were neutrophils present in the BALs of some mice but only in those groups sensitised and challenged with OVA and only in BALs sampled 24 hours after the last aerosol. Serum IgE Total serum IgE was significantly increased at day 21 (p < 0.001), 7 days after the second injection of OVA/Alum, compared to naïve mice (Fig. 6 ). In contrast, serum IgE levels at day 14, after a single injection, were not significantly elevated (p = 0.438) compared to naïve mice. The total serum IgE response to systemic sensitisation, in the absence of subsequent antigen aerosol challenge, peaked at day 22 and partially declined by day 27. However, this decrease was not statistically significant (p = 0.511). There was no further increase in the total serum IgE in mice that were sensitised and subsequently challenged with OVA aerosols compared to those that were only systemically sensitised (p = 0.842). Total serum IgE levels were not significantly greater in mice sensitised with PBS/Alum and challenged with OVA ( data not shown ). Figure 6 Total serum IgE obtained from time resolved fluorescence. Total IgE obtained from time resolved fluorescence assay of serum collected from systemically sensitised (i.p. OVA/Alum on day 0 and day 14) but not challenged with aerosolised antigen (white bars). The vertical bars represent total serum IgE from mice sensitised and challenged with either 1, 3 or 6 OVA aerosols. Serum samples from these mice were collected 24 (grey bars) and 48 (black bars) hours after the last aerosol. Data are expressed as mean ± SEM (n = 10). Two intraperitoneal injections of OVA/Alum were sufficient to induce increased levels total IgE by day 21 (p < 0.001) compared to naïve mice. Exposure to OVA aerosol challenges did not cause a further increase in total IgE (p = 0.842). Discussion Varying the number of aeroallergen challenges in a systemically sensitised murine model of allergic bronchopulmonary inflammation altered the degree and timing of hyperresponsiveness to inhaled MCh. A single OVA challenge increased airway sensitivity to inhaled MCh 24 hours after the challenge, while sensitivity remained elevated for 48 hours after three and six challenges. OVA challenge did not increase parenchymal sensitivity at any level. In contrast to sensitivity measurements, the maximum response to 30 mg.mL -1 MCh showed a variable pattern. A transient response was observed in both airway and parenchymal compartments after a single OVA aerosol. After 3 OVA aerosols significant increases were seen in the tissue compartment at 48 hours, while after 6 OVA aerosols an elevated response was seen in the airway and parenchymal compartments that persisted beyond 48 hours. There was a significant influx of inflammatory cells in the BAL in response to OVA aerosols, however, the presence of this inflammation did not always result in hyperessponsiveness to inhaled MCh. Murine models using 2 systemic allergen sensitisations followed by 3 aeroallergen challenges are prevalent in the literature ([ 20 , 29 - 31 ]) and have been reported to demonstrate airway hyperresponsiveness to MCh. However, these studies have used enhanced pause (Penh), which is derived from unrestrained barometric plethysmography, to measure changes in lung physiology. As Penh cannot differentiate between constriction in the airways and changes in the tissue compartment of the lungs, it is impossible to tell where the responses to MCh are localised, if indeed they are true physiological responses ([ 10 - 14 ]). In contrast, our study, using 2 systemic sensitisations and 1,3 or 6 challenges, has demonstrated clear airway, tissue or mixed compartment responses to methacholine which is dependent on the number of aerosol challenges delivered. In our hands, the more common model of 2 systemic sensitisations followed by 3 OVA challenges resulted in increased responsiveness to the maximum dose of MCh that was confined to the tissue compartment of the lung. This finding is consistent with a previous study by Tomioka et al . ([ 17 ]), which also used a forced oscillation technique to measure changes in lung mechanics in OVA sensitised and challenged mice. The fact that the response was confined to the tissues is of interest as the aim of these models is to mimic the human asthmatic condition, in which a significant portion of reactivity of the lungs is localised in the conducting airways ([ 19 ]). This work emphasises the importance of measuring bronchoconstriction with physiological techniques capable of compartmentalising responses within the lungs. By varying the antigen dose at challenge we have revealed a system with the potential to allow investigation of transient or prolonged responsiveness to MCh that is localised in the airways, tissues, or both. Further investigation is needed in order to understand the mechanisms that are influencing the site of responsiveness. Typically, most human studies measure MCh responsiveness in terms of sensitivity as they report the concentration of MCh required to produce a 20% fall in FEV 1 . We have shown that it is possible to determine sensitivity to inhaled MCh in mice and that only the airway compartment shows heightened sensitivity following allergic sensitisation and challenge. While increased maximal responses can be seen in both airway and parenchymal compartments, depending on which model is used, no increase in parenchymal sensitivity is seen with any of the models we used. As such, these findings reinforce the value of using lung function techniques that are capable of assessing airway and parenchymal mechanics separately. Total serum IgE was significantly elevated following systemic sensitisation but was not increased by aerosol challenge. There was, however, a tendency for total serum IgE to decline by day 27 in mice that were systemically sensitised but not challenged with OVA aerosols, compared to mice additionally exposed to 6 OVA aerosols. It is possible that if the study had been extended to include further exposure to antigen over subsequent days, a difference would have been detected between mice that were only sensitised and mice that were sensitised and challenged. Given that antigen specific IgE and other immunoglobulin subtypes were not measured in this study, further work is required to characterise the effect of dose of antigen at challenge on the development of antibody responses to OVA in mice. The protocol used in the present study induced significant eosinophilia after a single airway challenge. The degree of eosinophilia increased with increasing number of airway challenges. This finding is consistent with several previous studies using similar protocols to induce allergic inflammation in the lungs of mice ([ 20 , 29 - 31 ]). While the level of activation of the eosinophils was not measured in the present study, the 61% eosinophilia found after 6 OVA aerosols was much higher than those that are typically found in human asthmatics ([ 32 ]). Given the significant and prolonged parenchymal response to inhaled methacholine following 6 OVA aerosols and the level of eosinophilia present, it is likely that this model more closely parallels an allergic alveolitis ([ 33 ]) than the airway inflammation commonly seen in humans. In recent studies there has been some focus on the association, or lack thereof, between indicators of systemic sensitisation, such as the levels of serum antibodies, airway inflammation and AHR ([ 34 ]). In a review of the role of IgE in the induction of eosinophilic airway inflammation and AHR, Hamelmann et al . ([ 35 ]) concluded that systemic methods of sensitisation resulted in high levels of IgE and eosinophilic airway inflammation in BALB/c mice. In these models, AHR was determined to be dependent on eosinophils but not IgE. However, the results of our study, which uses a similar protocol to those reviewed by Hamelmann et al . ([ 35 ]), show that the presence of eosinophils did not always coincide with an increase in responsiveness to MCh. Three OVA aerosols resulted in a significant eosinophilia after 24 hours but an increase in the response to the maximum dose of MCh was not evident until 48 hours post challenge. In contrast, a single OVA challenge resulted in hyperresponsiveness to MCh that had resolved by 48 hours while the levels of eosinophils remained significantly elevated. The levels of total serum IgE were equivalent across all challenge doses suggesting that, while the presence of IgE may be necessary to initiate the allergic response, its presence at a particular measurement time point does not necessarily relate to the presence of hyperresponsivenss. Conclusions The findings of the present study demonstrate the significant impact of changing antigen challenge dose in a murine model of allergic bronchopulmonary inflammation. Given the variability of the inflammatory profile and characteristic responses observed in this study, it is clear that investigators must carefully characterise their allergen-driven murine models to ensure the model used contains the characteristic of interest. Future studies need to be directed at understanding the mechanisms that underlie airway and parenchymal hyperresponsiveness post antigen challenge. Authors' contributions GRZ carried out the animal studies and drafted the manuscript. CvG carried out the IgE analysis and assisted in the interpretation of results and editing the manuscript. PAS assisted in the interpretation of results and editing the manuscript. PGH assisted in the conceptualisation of the study and interpretation of the results. PDS and DJT assisted in the conceptualisation of the study, interpretation of the results and editing the manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521690.xml |
516028 | Cross-species global and subset gene expression profiling identifies genes involved in prostate cancer response to selenium | Background Gene expression technologies have the ability to generate vast amounts of data, yet there often resides only limited resources for subsequent validation studies. This necessitates the ability to perform sorting and prioritization of the output data. Previously described methodologies have used functional pathways or transcriptional regulatory grouping to sort genes for further study. In this paper we demonstrate a comparative genomics based method to leverage data from animal models to prioritize genes for validation. This approach allows one to develop a disease-based focus for the prioritization of gene data, a process that is essential for systems that lack significant functional pathway data yet have defined animal models. This method is made possible through the use of highly controlled spotted cDNA slide production and the use of comparative bioinformatics databases without the use of cross-species slide hybridizations. Results Using gene expression profiling we have demonstrated a similar whole transcriptome gene expression patterns in prostate cancer cells from human and rat prostate cancer cell lines both at baseline expression levels and after treatment with physiologic concentrations of the proposed chemopreventive agent Selenium. Using both the human PC3 and rat PAII prostate cancer cell lines have gone on to identify a subset of one hundred and fifty-four genes that demonstrate a similar level of differential expression to Selenium treatment in both species. Further analysis and data mining for two genes, the Insulin like Growth Factor Binding protein 3, and Retinoic X Receptor alpha, demonstrates an association with prostate cancer, functional pathway links, and protein-protein interactions that make these genes prime candidates for explaining the mechanism of Selenium's chemopreventive effect in prostate cancer. These genes are subsequently validated by western blots showing Selenium based induction and using tissue microarrays to demonstrate a significant association between downregulated protein expression and tumorigenesis, a process that is the reverse of what is seen in the presence of Selenium. Conclusions Thus the outlined process demonstrates similar baseline and selenium induced gene expression profiles between rat and human prostate cancers, and provides a method for identifying testable functional pathways for the action of Selenium's chemopreventive properties in prostate cancer. | Background Gene expression profiling, along with other methods to evaluate the global changes in genomes, provides the opportunity to understand whole scale changes present in human biology. Yet the sheer mass of data presented by these techniques often makes subsequent analysis difficult. Techniques such as gene expression profiling may result in the identification of hundreds if not thousands of differentially expressed genes that may be associated with the biological process, but may also represent noise related to the biological and technical variation. In an economic environment where limited resources are available for the follow-up and validation of potential target genes methods must be provided for the prioritization and sorting of data. Previous methods have relied heavily on the mapping of metabolic pathways or transcription factor binding sites [ 1 - 5 ]. These processes rely on the premise that the metabolic pathways associated with a given disease are well delineated, or that groups of proteins with very similar structural or functional design are involved in the disease process. In situations where these assumptions may not be true, alternative methods for the sorting of the data are needed. Here we demonstrate an alternative approach using comparative genomics and animal models of human prostate cancer to sort and identify genes involved in the response of prostate cancer cells to the proposed chemopreventive agent Selenium [ 6 , 7 ]. This process takes advantage of the continued sequencing of multiple animal genomes and the ability to produce gene expression profiles in multiple species. Through the use of these techniques one can leverage established animal models to identify genes associated with human disease processes, as is demonstrated here with the identification of Insulin-like growth factor-2 Binding protein 3 (IGFBP3) and retinoid-X-receptor alpha (RXRalpha). Results Generation of common genes and homologs Sequence validated gene libraries for both the rat and human DNAs were obtained from Research Genetics (Huntsville, AL), and were supplemented with additional DNA samples obtained from the University of Iowa rat clone sequencing program [ 8 ]. The majority of the rat DNAs, and a subset of the human DNAs were resequenced by Dr. J. Quackenbush at TIGR through a joint Program in Genomic Applications consortium. The GeneBank accession numbers for the 19,200 individual human or rat clones present in the recent slide printings were used to query the NCBI Unigene database to return the associated Unigene IDs. Unigene IDs were returned for virtually all identified clones, and were placed in an Oracle database where they were compared with the downloaded NCBI Homologene dataset (build 106) of rat, mouse, and human homologues. Of the 19,200 clones, 5740 genes were identified with homologues present on both the rat and human slides. This homologue set was used for the subsequent comparisons across species. Similar global and prostate gene expression profiles between rat and human prostate cancer cell lines We have sought to compare the rat and human prostate cancer transcriptomes in an effort to judge the degree of similarity between the two cell types. Because the use of differentially expressed genes would bias the comparison by eliminating the majority of genes that do not show any difference, we used the absolute level of expression for each gene and compared the rat and human genes for significant differences in absolute expression levels. In order to derive the absolute level of expression for individual genes in human or rat prostate cancer cells we used expression values derived from the associated self-self hybridizations performed for each cell line. The experiments were facilitated by the use of slides that have been quality controlled for the quantity of spotted target DNA through the use of a FITC label third dye [ 9 ]. These slides were subsequently imaged for FITC fluorescence and sorted based on the similar amounts of target DNA present on each slide [ 10 ]. Using the third dye quality control correlation coefficients of greater than 0.80 are routinely achieved between slide replicates [ 9 ]. In this manner comparisons of bound hybridized probe can be made across slides with a degree of confidence. RNA samples from cells were harvested, labeled, and homotypically hybridized to establish the baseline level of consistency within the hybridizations. Performing slice analysis on the normalized homotypic gene expression data across all the self-self hybridization slides within a species and retaining genes that demonstrated consistent expression patterns within two standard deviations of the mean expression value was performed to remove a degree of error from the technical replicates. Using the third dye as a baseline for comparison, these common expressed genes were then broken down into their component Cy3 or Cy5 expression vectors and used to build the transcriptomes for each gene using their absolute expression values. These transcriptomes were then used to compare expression values between the rat and human cell lines. These genes were annotated and gene homologues identified from the NCBI Homologene[ 11 ] dataset of rat-human homologues. Thus from a dataset of 5740 homologues, 2883 genes were found that were present within this experimental dataset and expressed in both the rat and human prostate cancer cell lines, and thus could be used for comparative genomics. These samples were processed using the Multiexperiment Viewer mircoarray statistical analysis and visualization program developed by TIGR [ 12 ]. Files were loaded and visualized for comparison across the 2883 common expressed genes in a self-organizing tree algorhythm [ 13 ] (figure 1 ) and analyzed for similarities in global expression patterns. The hierarchical clustering in self-organizing trees failed to demonstrate a pattern of clustering between species. T-test analysis [ 12 , 14 ] between the human and rat cell lines identified 58 genes (2%) which demonstrated significantly different expression patterns between species (p < 0.01 with Bonferroni correction). Thus in these comparisons, 2826 genes, or 98% of the genes examined, failed to demonstrate a statistically significant difference in expression between the human and rat prostate cancer cell lines. Using principle components analysis (figure 2 , [ 12 ]) these studies can be visualized, and demonstrate while there is some clustering of the rat and human prostate cancer cell lines, the differences are not significant. Thus when comparing gene expression patterns in rat and human cell lines one will detect significant species-specific differences in expression in 1 out of every 50 genes, with the majority of the genes demonstrating similar expression patterns. Figure 1 Gene expression profiles for human and rat prostate cancer cells. Clustering of the expressed genes in the human (LNCAP, DU145, PRO4, LN4, and PC3 derivatives) and rat (AT3, MatLyLu, and PAIII) prostate cancer cell lines based on the common homologs as defined within to NCBI Homologene database. Raw data files are available for review from the corresponding author. Figure 2 Principal Components Analysis of Rat and Human Prostate Cancer Cell Lines. There is a clustering of the human (Pro4-purple, LN4-dark-blue, PC3S-light blue, PC3US-yellow) and rat (MatLyLu-red, AT3-magenta, PAIII-green) prostate cancer cell lines in the same quadrant. The degree of separation within the quadrant was not significant by T-testing. Each sample is presented in duplicate based on independent Cy3 and Cy5 vector profiles. The presence of a large quantity of genes whose expression may be related to general cellular functions, as opposed to prostate specific metabolism, could infuse a significant amount of homogeneity to the data. In the presence of such homogeneity it may be impossible to identify the true differences that are related to prostate cellular function, and thus the perceived similarities may be artifactual. To address this issue we sought to repeat the analysis using only prostate related genes. To generate a list of such genes we used cDNAs in eight normal human prostate cDNA libraries present in the NCI Cancer Genome Anatomy Project [ 15 ]. Generation of a list of common genes proved impossible, as the combination of more than four of the cDNA lists resulted in the number of common genes being reduced to zero. A similar result was obtained when one attempted to generate a list of commonly expressed genes across multiple different cancer cDNA libraries. As an alternative approach we developed a list of 12,008 expressed genes were identified based on their presence in at least one of the eight normal human prostate cDNA libraries. The human Unigene IDs for each of the expressed genes were then used to identify the associated rat homologues from Homologene [ 11 ] and yielded 2,269 homologous rat genes (18.9%), of which 1,319 (58.1%) had associated prostate cancer gene expression data. These 1,319 prostate expressed genes were then used to repeat the comparative genomics. Similar visual and clustering results were identified for the prostate transcriptomes. T-test analysis [ 12 , 14 ] between the human and rat cell lines identified 30 prostate expressed genes (2%) which demonstrated significant differential expression between species (p < 0.01 with Bonferroni correction, while 1,289 genes (98%) failed to demonstrate a significant difference in expression across species. Thus even when only prostate expressed genes are considered, similar results were obtained. Between the rat and human prostate cancer cell lines the patterns of expression are similar for 49 of 50 genes examined. Comparison of global and prostate specific differential gene expression profiles between rat and human prostate cancer cell lines treated with selenium While global gene expression profiles appear to be similar between rat and human prostate cancer cell lines one wonders whether the response to specific physiologic stimuli may elicit similar transcriptional changes. If so, one may be able to infer a degree of homology in their biological response to the stimuli. This has already been observed on a physiological level for the rat models of prostate cancer. For example, rat and human prostate cancers respond very similarly to chemotheraputic and environmental agents including hormonal agents (both respond), cyclophosphamide (neither respond), high fat diets (increased incidence), and soy isoflavones (decreased incidence) [ 16 - 22 ]. In an effort to evaluate these similar biological responses we have compared the transcriptomes between rat and human prostate cancer cell lines treated with the proposed prostate cancer chemopreventive agent Selenium. Samples from the human PC3 and rat PA-III cell lines were treated with Selenium and examined for differential gene expression profiling. These two cell lines were chosen based on their similar biologic characteristics, as both cell lines were derived from androgen independent metastatic tumors, and thus represent tumors with similar biologic potential [ 23 , 24 ]. The cells were treated with twenty-five micromolar Selenium for either 6 hours or 5 days, to identify both immediate changes in gene transcription or changes related to the long term exposure to Selenium. Due to our interest in prostate cancer we have attempted to choose a form and concentration of Selenium that would be reflected in the ongoing prevention trials such as the SELECT prostate cancer prevention trial [ 25 , 26 ]. In this trial patients receive Selenium in the form of Selenized baker's yeast. Previous HPLC and electrospray mass spectroscopy studies have demonstrated that 85% of the Selenium in yeast is present as selenomethionine [ 27 ]. Selenomethionine has previously been used in in-vitro studies of prostate cancer cells[ 28 , 29 ]. These studies demonstrated an inhibition of prostate cancer cell proliferation over a broad range of concentrations, while an IC50 and/or decreased expression was seen at concentrations above 70 micromolar selenomethionine. To avoid the general effects of cell inhibition or cell death while focusing on the effect of Selenium we chose a lower concentration of 25 micromolar selenomethionine. These changes, while not resulting in increased cell death, did cause decreased cell division and increased doubling time in both species (data not shown). Common rat and human homologous genes demonstrating differential expression by greater than two standard deviations were identified and included 1123 genes after 6 hours and 1053 genes after 5 days of exposure to Selenium. When the expression patterns of these genes were compared across species by T-test and principle component analysis as outlined above 713 genes (25%) were found to have statistically significant differences in expression between species (p < 0.01 with Bonferroni correction). Thus when comparing rat and human samples, while the majority of the gene expression changes are similar, in at least one in four genes (p = 0.75) one can detect significant species specific differences in expression alteration when cells are treated with Selenium. Yet similar physiologic changes (decreased cellular proliferation, increased cell death) were observed in both species. These changes represent the desired physiologic changes one would expect for the chemopreventive effects of Selenium, and could be dissected by examining the common transcriptional changes seen in both species with respect to Selenium. Combined differential expression patterns for selenium responsive genes identify common gene pathways Because some of the differences in the rat and human prostate cancer cell line transcriptomes may be related to confounding variables such as culture methods, cell passage number, or time in culture, an effort was made to focus on genes that are common, and as such may define the similar Selenium based cell proliferative changes. The subsets of 1123 and 1053 differentially expressed genes (6 hours and 5 days respectively) were analyzed for genes that demonstrate similar changes in expression with respect to Selenium across species. Of these differentially expressed genes, 291 and 309 demonstrated up-regulation in rat and human cells at 6 hours and 5 days respectively. Likewise, 261 (6 hours) and 216 (5 days) demonstrated down-regulation in the presence of Selenium. When these subsets were further analyzed to identify genes with similar levels of up or down-regulation (defined as ratio differences within 0.2 units of each other) 81 genes were identified at 6 hours and 73 at 5 days (table 1-see additional file 1 ). These genes included 40 ESTs or genes with limited associated data, and 90 defined genes with associated gene data. Twenty-four of the genes were common to Selenium treatment at both 6 hours and 5 days. Additional information related to these genes was obtained using the GeneInfo data mining tool. This tool was developed by the authors (MWD, XW, HL, GZ) to allow for the rapid identification of supplemental data from the biomedical literature related to genes of interest. In brief, the tool allows one to cut and paste a list of genes based on either Unigene or Genebank IDs and search PubMed for associated references based on annotations of the associated gene names. Additional search terms can be stipulated by the user based on their knowledge of the biological process or in response to results received from the previous search. Results are returned in a table that lists the number of references that met the search criteria and provides a hyperlink to the associated references for either downloading or viewing. In this way the user is allowed to direct queries in an open manner based on their own experience or unpublished data. In this manner searches were conducted using the list of genes and the search terms "prostate cancer", "Selenium", and "apoptosis" (table 1-see additional file 1 ). IGFBP3 and RXR-alpha are expressed in the prostate, induced by selenium, and downregulated in prostate cancer Of the 154 genes identified with similar cross-species differential expression changes with respect to Selenium, two genes were identified that had unique features based on their associated references and interrelated functions. These genes, IGFBP3 and RXR-alpha were both up-regulated with respect to Selenium and could be used to suggest a model for Selenium action in prostate cancer. PXR-alpha is upregulated in both rat and human prostate cancer cells at 5 days in response to Selenium. Likewise, IGFBP3 is upregulated after six hours of Selenium treatment in both species. These two genes both contained Medline references with respect to prostate cancer, but had not yet been implicated in Selenium action. Western blotting performed on the human prostate cancer cell line PC3 with respect to Selenium validated the bioinformatically identified expression data (figure 3 ). To confirm the role of these two proteins in the prostate immunohistochemical studies on prostate cancer tissue microarrays were performed to identify IGFBP3 and RXR-alpha in both normal, nodular hyperplasia (benign prostatic hypertrophy), high grade prostatic intraepithelial neoplasia (HGPIN), invasive carcinoma, and metastatic prostatic carcinoma (table 2 ). These studies demonstrate that both IGFBP3 and RXR-alpha are expressed in the normal human prostatic epithelium (figure 4 , table 2 ). IGFBP3 is also expressed in the prostatic basal cells. Patterns of expression were predominantly nuclear, a finding that has been described for both proteins [ 30 ]. In addition, staining for IGFBP3 was also noted in the prostatic stroma, consistent with IGFBP3's associated function as a secreted protein. Decreased levels of IGFBP3 was noted in prostatic cancers when compared to normal prostate epithelium (p = 0.0044). Along with this decreased expression there was a distinct shift in the protein localization nuclear to cytoplasmic was observed (p < 0.00001), and in cases where expression was still present, there were decreased numbers and intensity of cell staining. IGFBP3 expression was similar in HGPIN, invasive carcinoma, and metastatic carcinoma. The level and pattern of IGFBP3 expression in nodular hyperplasia was similar to that seen in normal prostate tissues, and significantly different from the expression seen in cancer samples (p = 0.0036 and p < 0.00001 respectively). RXR-alpha expression was also significantly downregulated in prostate cancer when compared to normal prostate epithelium or nodular hyperplasia (p < 0.0001), and was similar to that seen in HGPIN and metastatic carcinoma. RXR-alpha expression was consistently nuclear in the samples studied, and while the intensity of staining was similar, in the remaining positive cancer cases there were decreased numbers of cells staining (8.6 +/- 12.6% in malignant epithelium vs 20.0 +/- 25.5% in normal epithelium). Figure 3 Expression of IGFBP3 and RXR-alpha with respect to Selenium. Western blotting reveals an induction of RXR-alpha or IGFBP-3 protein after Selenium treatment of human PC3 prostate cancer cells (arrows, upper row). Western blotting of immunoprecipitations from rat PAIII cells (bottom row) reveal RXR-alpha in immunoprecipitated IGFBP3 extracts (right panel) and IGFBP-3 in immunoprecipitated RXR-alpha extracts confirming and extending the reported interactions between the human proteins[40]. Table 2 Expression of IGFBP3 and RXRalpha in Prostatic Epithelium Normal Prostate Nodular Hyperplasia HGPIN Prostate Cancer Metastatic Cancer IGFBP3 Positive cases 105 62 49 202 25 Negative cases 5 1 9 36 8 Statistics (comparison) p = 0.0036 (cancer) N.S. (cancer) p = 0.0044 (normal) N.S. (cancer) IGFBP3 Intensity (avg+/-std) 2.47 +/- 0.70 2.49 +/- 0.65 2.57 +/- 0.82 2.74 +/- 0.56 2.79 +/- 0.49 Percentage cells (avg+/- std) 8.3 +/- 13.5 7.5 +/- 12.5 8.8 +/- 15.2 4.4 +/- 6.6 8.5 +/- 12.6 Nuclear cases 92 59 40 94 8 Cytoplasmic cases 22 6 18 152 11 Statistics (comparison) p < 0.00001 (cancer) p = 0.065 (cancer) p < 0.00001 (normal) N.S. (cancer) RXRalpha Positive cases 92 58 35 112 16 Negative cases 10 3 31 125 19 Statistics (comparison) p < 0.00001 (cancer) N.S. (cancer) p < 0.00001 (normal) N.S. (cancer) RXRalpha Intensity (avg+/-std) 2.73 +/- 0.51 2.78 +/- 0.50 2.83 +/- 0.38 2.76 +/- 0.49 3 +/- 0 Percentage cells (avg+/- std) 20.0 +/- 25.5 23.2 +/- 25.7 8.4 +/- 12.5 8.6 +/- 12.6 4.2 +/- 4.6 Nuclear cases 92 58 35 107 16 Cytoplasmic cases 2 0 6 9 0 Statistics (comparison) N.S. (cancer) N.S. (cancer) N.S. (normal) N.S. (cancer) Figure 4 Expression of IGFBP3 and RXRalpha in human prostate tissues. Immunohistochemical staining for IGFBP3 is present as brown staining in normal prostate (A) and prostate cancer (C). Similarly RXRalpha expression is present in normal prostate (B) and lost in prostate cancer (D). All images recorded at 100× magnification. Discussion Leveraging cross-species bioinformatics in the prioritization of gene data Through the use of cross-species comparisons of the number of differentially expressed genes to be examined after 6 hours and 5 days of Selenium treatment was dropped from 9453 and 7768 to 1123 and 1053 respectively, an 87–89 percent reduction of the sample size. Even with the use of multiple timepoints, the number of differentially expressed genes was only reduced in a single species study to 5934, less than half. By using comparative genomics the final dataset was reduced to 154 genes, providing a greater than 100 fold enrichment of the data. Thus by leveraging the additional biological species the ability to reduce the final analysis pool was substantial. This process only works if the species used have biological relevance to the disease in question. The choice of rat prostate cancer cell lines was made based on their use as an animal model for the study of prostate cancer [ 31 ]. The animal systems have been extensively used in the study of hormonal carcinogenesis, and in particular have been of value as a model of environmental and dietary effects on prostate cancer [ 18 - 20 ]. Previous studies have identified similar effects of rat animal models and prostate cancer cell lines to soy based diets [ 17 - 19 ], high fat diets [ 20 - 22 ], hormonal chemotherapeutics (Pollard, personal communication) and standard chemotherapy [ 32 , 33 ]. While comparative gene expression profiling has been performed, this has usually been through cross-species hybridizations to leverage RNA studies in species where sufficient expressed transcripts in a given species have not been identified for the production of species-specific gene expression slides, in particular for microbial genomes [ 34 - 37 ]. Thus the approach taken here leverages the production of species-specific gene expression profiles along with the increasing amount of gene homolog data generated by the sequencing of additional animal genomes. It is expected that with future genome efforts additional cross-species studies will be possible that leverage the knowledge of additional animal models in the study of disease. Similarities in prostate cancer transcriptomes across species For both overall and prostate expressed genes, we have failed to identify a significant difference in the transcriptomes between rat and human prostate cancer cell lines. This general similarity in transcriptomes may be due to the inherent biological similarities of the cell lines and/or their underlying biological origin. While the studies sought to utilize prostate cancer cell lines with similar biological potentials (established cell lines all derived from metastases) the degree of diversity present within the samples may account for some of the residual differences still identified. In addition, the extended period of time that these cell lines have been used has allowed for the continued in-vitro evolution of the cells, and could possibly extend those genomic differences. Yet the common clustering of the rat and human cell lines together suggests there are still significant similarities in their biological potential. This is also demonstrated by the similar biological potential of the cell lines when treated with a given stimulus, in this example Selenium. This parallels the similar physiological properties observed in the rat models of human prostate cancer. Based on these features we demonstrate that it is possible to identify functionally significant genes related to Selenium response by using comparative genomics. These findings also support the use of animal models in the study of human prostate cancer by suggesting that there is enough inherent genomic similarity that valuable insights may be gained from animal systems. Comparative genomics identifies functionally significant genes with respect to selenium chemoprevention A true test of the profiling method is the identification of genes that have a functional significance to the experimental system. In this case we have identified a series of genes, which when examined with additional data mining techniques, identifies genes with associated roles related to apoptosis (IGFBP3, RXRalpha, dynamin-2), antioxidant protection (selenoprotein N, peroxiredoxin I, zinc metalloprotease, glutathione S transferase), cell cycle (CDC26-anaphase promoting complex, kinetochore associated protein), and protein balance (proteasome subunit beta-4, ubiquitin conjugating enzyme). In addition, the ability to sort the identified genes by their associated biomedical literature allowed the focus to shift to IGFBP3 and RXRalpha. Retinoids, through the retinoid X receptor, have been shown to induce the expression of IGFBP3 [ 38 ]. In concert these two proteins act to induce apoptosis in cancer cell lines [ 39 ]. In particular, recent data has shown that these proteins work in synergy to enhance apoptosis in prostate cancer, and that there is a physical interaction between these two proteins in prostate cancer cells[ 40 ]. Further validation and confirmatory data is presented here that demonstrates the selenium induced expression and interaction between both RXRalpha and IGFBP3 in prostate cancer cells, along with their expression in normal prostate epithelium and subsequent down-regulation in malignant prostatic epithelium. This allows one to pose a model by which the restoration of IGFBP3 and RXRalpha levels by Selenium treatment may lead to the disruption of prostate tumorigenesis. This model is testable, and if validated, would present not only a mechanism by which Selenium may exert its effect, but provide a biomarker for assaying the effect of Selenium supplementation in the ongoing prostate cancer prevention clinical trials. Conclusions Using gene profiling on highly controlled spotted cDNA arrays we have demonstrated that similar baseline and selenium induced gene expression profiles can be identified between rat and human prostate cancer cells. This has allowed us to filter our gene expression data to identify genes whose transcriptional response to Selenium is similar across species, and by so doing focus our discovery process on specific common physiologic pathways. Two such proteins, RXR-alpha and IGFBP-3, which may be located in a common pathway, have been identified as dysregulated in human prostate cancers. This provides further support that the cross-species methods employed here can identify genes with roles in human prostate cancer. Methods Cell culture and selenium treatment Cell lines were received from ATCC, Rockford, MD, (LNCap, DU-145, MatLyLu, AT3), from Drs. Paul Lindholm and Andre Kadjacsy-Balla (LN4, Pro4, PC3, PC3-NI(PC3US), PC3-I(PC3-S)), or Dr. Morris Pollard and Mark Suckow (PA-III). These cells were cultured in RPMI (DU-145) or DME medium supplemented with 10% fetal calf serum, 10 mM glutamine, and 10 mM sodium pyruvate, and passaged 1:8 or 1:10 when the cells reached 70–80% confluence with trypsin-EDTA. For the Selenium studies PC3 or PAIII cells from a single cell stock were seeded at 1 × 10EE4 cells per ml and grown to 50% confluence at which time the culture medium was changed to either standard growth medium (above) or medium supplemented with twenty-five micromolar Selenium (Seleno-DL-methionine, Sigma cat# S3875, St. Louis MO). The cells were then cultured for an additional 6 hours or 5 days. Cells that reached 80% confluence prior to the five day timepoint were split using trypsin-EDTA and replated in either control or selenium-containing medium for the duration of the experiment. Cells were monitored for viability and cell growth with parallel growth curves conducted in triplicate, this data demonstrated the previously described [ 41 , 42 ] decrease in cellular proliferation (data not shown) observed in the presence of Selenium. RNA isolation and quantitation RNA was isolated from cells using Trizol (Invitrogen cat # 15596018, Carlsbad, CA) and subsequently examined for quality using agarose gel electrophoresis and Gelstar nucleic acid stain against known RNA standards and failed to demonstrate significant degradation based on the presence of high molecular weight RNA species, and intact 28s and 18s ribosomal RNA bands. DNA library preparation and amplification Sequence-verified rat and human libraries (Research Genetics, Huntsville, AL, and University of Iowa cDNA clone set, IA), consisting of 41,472 human clones and 36,000 rat clones were used as a source of probe DNA. A subset of 200 randomly selected clones were chosen from these libraries, resequenced locally, and demonstrated clone accuracy of 92%. We have opted to reformat libraries from 96 to 384-format for culture growth/archiving, PCR, purification, and printing. This has reduced the number of plates of our 41,472 human clone library from 432 to a more manageable 108, and the rat clone library from 375 to 94. The library was reformatted and subsequently manipulated using slot pin replicator tools (VP Scientific, San Diego, CA). Cultures were grown in 150 ul Terrific Broth (Sigma, St. Louis, MO) supplemented with 100 mg/ml ampicillin in 384 deep-well plates (Matrix Technologies, Hudson, NH) sealed with air pore tape sheets (Qiagen, Valencia, CA) and incubated with shaking for 14–16 hr. Clone inserts were amplified in duplicate in 384-well format from 0.5 μl bacterial culture diluted 1:8 in sterile distilled water or from 0.5 μl purified plasmid (controls only) using 0.26 μM of each vector primer {SK865 5'-fluorescein-GTC CGT ATG TTG TGT GGA A-3' and SK536: 5'-fluorescein-GCG AAA GGG GGA TGT GCT G-3'} (Integrated DNA Technologies, Coralville, IA) in a 20 μl reaction consisting of 10 mM Tris-HCl pH8.3, 3.0 mM MgCl 2 , 50 mM KCl, 0.2 mM each dNTP (Amersham, Piscataway, NJ), 1 M betaine, and 0.50 U Taq polymerase (Roche, Indianapolis IN). Reactions were amplified with a touchdown thermal profile consisting of 94°C for 5 min; 20 cycles of 94°C for 1 min, 60°C for 1 min (minus 0.5° per cycle), 72°C for 1 min; and 15 cycles of 94°C for 5 min; 20 cycles 94°C for 1 min, 55°C for 1 min, 72°C for 1 min; terminated with a 7 min hold at 72°. PCR reactions analyzed for single products by 1% agarose gel electrophoresis analysis. Products from replicate plates were pooled and then purified by size exclusion filtration using the Multiscreen 384 PCR filter plates (Millipore, Bedford, MA) to remove unincorporated primer and PCR reaction components. Forty wells of each 384-well probe plate were quantified by the PicoGreen assay (Molecular Probes, Eugene, OR) according to the manufacturers instructions. After quantification, all plates were dried down, and reconstituted at 150 ng/μl in 3% DMSO/1.5 M betaine. Array slide fabrication A single printing array containing 19,200 elements (human) or 2 arrays of 9,600 (rat), were printed on poly-L-lysine coated slides prepared in-house (1–2 arrays/slide) as previously described [ 9 ]. Printing was conducted with a GeneMachines Omni Grid printer (San Carlos, CA) with 16 or 32 Telechem International SMP3 pins (Sunnyvale, CA) at 40% humidity and 22°C. To control pin contact force and duration, the instrument was set with the following Z motion parameters, velocity: 7 cm/sec, acceleration: 100 cm/sec 2 , deceleration: 100 cm/sec 2 . All slides were post-processed using the previously described nonaqueous protocol[ 9 ]. Slide coating was performed as described previously [ 43 ]. Image files on all arrays were collected after blocking (fluorescein), and again after hybridization (Cy3 and Cy5) with a ScanArray 5000 (GSI Lumonics, Billerica, MA). Experimental design and bioinformatics based data analysis The experimental design utilized two biological replicates for each comparison with each replicate incorporating a Cy3/Cy5 dye flip. In addition, self-self hybridizations were performed for each sample to ensure experimental accuracy and evaluate expression bias. Comparisons were organized in a loop design for either human or rat prostate cancer cell lines, or were run as two-sample comparisons of baseline untreated control and Selenium treated cells. Array image TIFF files were analyzed with Gleams software (Nutec Sciences, Atlanta, GA). Additional TIFF file analysis, data normalization, clustering, and principle components analysis was performed using the Spotfinder, MIDAS and MultiExperiment Viewer Software from The Institute for Genomic Research (TIGR, Rockville, MD, [ 44 ], [ 12 ]) and used default values set in the MCW Practical Guide to TIGR Software Use (M. Datta, unpublished). In brief, image expression data was used as channel intensity minus background and intensity thresholds were set at a value of 300. Images were analyzed as dye flip pairs normalized using MIDAS with LocFit based LOWESS normalization and slice analysis set at two standard deviation cutoffs and a sample data population of 500 [ 45 ]. Samples were then averaged across two dye flip replicate pairs with removal of zero/dropped values using locally developed averaging software from the BEAR microarray suite (M. Datta, submitted). These final averaged values were subsequently annotated using the BEAR suite annotator and used for pattern identification and correlation with gene homologs. Homologous genes were identified from the NCBI homologene database ftp files and parsed using local scripts and databases present in the Bioinformatics Program,[ 46 ]. Additional data mining to identify references in the biomedical literature associated with specific genes and user chosen search terms was performed using the locally developed GeneInfo data tool (M. Datta, submitted). Raw data files, along with analyzed data subsets are available for use and study and can be obtained via a secure ftp site after contacting the corresponding author mdatta@mcw.edu . Protein purification, western blotting, and immunoprecipitation Protein extracts were prepared and immunoprecipitations and/or western blots made from five day twenty-five micromolar Selenium treated or control PC3 or PAIII prostate cancer cell lines as described previously[ 47 ]. In brief, ten micrograms of total protein were run on pre-cast 12% reducing SDS PAGE gels (Bio-Rad Labs, Hurcules, CA) and transferred to PVDF membranes. After blocking with caseine blocking buffer (Bio-Rad Labs, Hurcules, CA) the PVDF membranes were incubated with either anti-RXR-alpha or anti-IGFBP-3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 200 μg/ml dilutions, washed, and incubated with anti-rabbit secondary antibody (2 μg/ml) and developed with ECL Chemiluminescence (cat. RPN2108, Amersham Biosciences, Piscataway, New Jersey). Immunoprecipitations were carried out using 200 microgram samples of total cellular protein, which after preclearing with protein A agarose beads was sequentially incubated with either anti-RXR-alpha (1 μg/ml) or anti-IGFBP-3 (1 μg/ml) antibodies, washed, incubated with anti-rabbit protein A agarose beads, washed, and the protein pellet western blotted with the complimentary antibody (anti-IGFBP-3 or anti-RXR-alpha, respectively), and developed with ECL Chemiluminescence. Tissue microarray production, immunohistochemistry, and analysis After expedited institutional review board approval normal prostate tissues and prostate cancer samples were obtained from de-identified discarded patient specimens. The formalin-fixed paraffin embedded specimens were prepared as 5 micron sections. Tissue microarrays were prepared from donor tissue blocks as 0.6 mm cores in 12 (4 × 4) or (5 × 5) grids with between 192 to 300 samples and used in the preparation of 5 micron sections. Immunohistochemistry was performed using primary rabbit polyclonal antibodies to the insulin-like growth factor binding protein 3 (IGFBP3, 1:300, Santa Cruz Biotechnology, Santa Cruz, CA), or retinoic-X-receptor alpha (RXR-alpha, 1:800, Santa Cruz Biotechnology, Santa Cruz, CA) using methods previously described [ 48 , 49 ]. In brief, endogenous peroxidase from deparaffinized sections were blocked with Methanol/Acetic acid, and after treatment with blocking serum (ABC kit, Pierce Biotechnology, Rockford, IL) samples were incubated for 30 minutes with either anti-IGFBP3 (1:300) or anti-RXRalpha (1:600). Sections were subsequently washed, and incubated with anti-rabbitt secondary antibody conjugated to horseradish peroxidase and counterstained with Mayers hematoxalin. Antigen retrieval (90 C waterbath for 10 minutes) was used for RXRalpha. Positive controls for each antibody included nuclear staining in Sertoli cells [ 50 ] and lymphocytes[ 51 ]. Positive staining was recorded and scored on a 0–2 scale (0 = no staining, 1 = staining that does not obscure the hematoxalyn counterstain, 2 = staining that obscures the hematoxalyn counterstain). Evidence of positive staining was recorded as presence of staining (yes/no) or percent of epithelial or basal cells staining (number of cells staining over total number of cells). Patterns of staining (nuclear, cytoplasmic, membranous, diffuse extracellular) were also recorded. All samples were analyzed and recorded by two separate personnel, including a trained urologic pathologist (MWD, BM). Statistical analysis was performed using Chi-squared probability analysis. Abbreviations None declared. Authors contributions M.W.D. was responsible for the conception and implementation of this project in association with P.J.T., M.S., and M.P. H.L., X.W., and G.Z. were actively involved in the programming, database construction, and testing of the software. M.S. and M.H. were responsible for spotted cDNA construction, hybridization, and experimental analysis along with M.W.D. Cell culture, western blots, immunoprecipitations, and selenium treatments were performed by M.S. with assistance by B.M. Tissue microarray staining and analysis was performed by M.W.D., R.D., T.B., and B.M. All the authors reviewed and accepted the final version of the paper. Supplementary Material Additional File 1 Table 1, Word document, Table of the genes identified in the selenium gene expression studies. Click here for file | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516028.xml |
549538 | Changes in lipids over twelve months after initiating protease inhibitor therapy among persons treated for HIV/AIDS | Background Protease inhibitors are known to alter the lipid profiles in subjects treated for HIV/AIDS. However, the magnitude of this effect on plasma lipoproteins and lipids has not been adequately quantified. Objective To estimate the changes in plasma lipoproteins and triglycerides occurring within 12 months of initiating PI-based antiretroviral therapy among HIV/AIDS afflicted subjects. Methods We included all antiretroviral naïve HIV-infected persons treated at St-Paul's Hospital, British Columbia, Canada, who initiated therapy with protease inhibitor antiretroviral (ARV) drugs between August 1996 and January 2002 and who had at least one plasma lipid measurement. Longitudinal associations between medication use and plasma lipids were estimated using mixed effects models that accounted for repeated measures on the same subjects and were adjusted for age, sex, time dependent CD4+ T-cell count, and time dependent cumulative use of non-nucleoside reverse transcriptase inhibitors and adherence. The cumulative number of prescriptions filled for PIs was considered time dependent. We estimated the changes in the 12 months following any initiation of a PI based regimen. Results A total of 679 eligible subjects were dispensed nucleoside analogues and PI at the initiation of therapy. Over a median 47 months of follow-up (interquartile range (IQR): 29–62), subjects had a median of 3 (IQR: 1–6) blood lipid measurements. Twelve months after treatment initiation of PI use, there was an estimated 20% (95% confidence interval: 17% – 24%) increase in total cholesterol and 22% (12% – 33%) increase in triglycerides. Conclusions Twelve months after treatment initiation with PIs, statistically significant increases in total cholesterol and triglycerides levels were observed in HIV-infected patients under conditions of standard treatment. Our results contribute to the growing body of evidence implicating PIs in the development of blood lipid abnormalities. In conjunction with the predominance or men, high rates of smoking, and aging of the treated HIV-positive population, elevated lipoproteins and triglycerides may mean that patients such as these are at elevated risk for cardiovascular events in the future. | Introduction Abnormalities in the lipid metabolism of persons infected with human immunodeficiency virus (HIV), potentially induced by the disease itself and the medications used for treatment, were first reported in the early 1990s[ 1 ]. Reductions in high- (HDL) and low-density lipoprotein (LDL) cholesterol were observed amongst persons infected with HIV and increases in triglycerides were observed among persons with AIDS[ 1 ]. Following the introduction of protease inhibitors (PI), morphological changes in fat distribution were reported. This was followed by numerous reports of metabolic disturbances, including glucose and lipid abnormalities presenting as insulin resistance, impaired glucose tolerance, hyperglycemia, type 2 diabetes mellitus [ 2 - 5 ], elevated serum triglycerides, LDL and very low density lipoprotein cholesterol, apolipoprotein B, E, and lipoprotein(a) [ 2 - 4 , 6 ]. The combination of metabolic disturbances and morphological changes are now described as the HIV-related "lipodystrophy syndrome"[ 2 , 3 ]. Up to one-half of subjects treated with PIs have shown elevated levels of triglycerides, total cholesterol (TChol), LDL, insulin, and fasting glucose [ 7 - 10 ]. The onset of metabolic changes appears to occur soon after initiation of treatment, often as quickly as within several weeks[ 11 ]. The high prevalence of disturbances of lipoproteins and triglycerides, the rapidity of their onset, and large changes that have been observed in randomized trials have led to concern regarding the potential impact of PIs on the cardiovascular health of persons with HIV/AIDS. However, the direction and magnitude of changes in plasma lipoproteins and triglycerides induced by PIs has yet to be quantified in an observational setting. The aim of this study was to quantify the magnitude of change in lipoprotein and triglyceride levels over twelve months following any initiation of PI-based treatment in a cohort of subjects treated for HIV/AIDS in a large tertiary care institution. Methods We included all antiretroviral naïve HIV-infected persons treated at St-Paul's Hospital, British Columbia (BC), Canada, who initiated use of PIs between August 1996 and January 2002. Plasma lipoprotein and triglyceride levels were obtained from measurements taken over the course of regular monitoring. Longitudinal effects of the impact of PIs on lipoproteins and triglycerides were estimated using statistical models that accounted for correlation due to repeated measurements on the same individuals. Study Setting and Population In BC, antiretroviral drugs have been centrally distributed at no cost to eligible HIV-infected individuals since 1986[ 12 , 13 ]. In October 1992, the HIV/AIDS Drug Treatment Program became the responsibility of the BC Centre for Excellence in HIV/AIDS. Since December 1996, the mainstay of treatment for HIV/AIDS has been highly active antiretroviral therapy (HAART) including two nucleosides and either a PI or a non-nucleoside reverse transcriptase inhibitor (NNRTI). Typically, HIV-infected subjects receiving antiretroviral therapy are monitored by physicians at intervals no longer than three months at which time prescriptions are renewed or modified based on clinical and laboratory parameters, and necessary laboratory tests are conducted. This research received ethical approval from the Institutional Review Board of Providence Health Care in BC. Exposure to antiretroviral therapy for HIV/AIDS At the BC Centre for Excellence in HIV/AIDS, records of CD4+ T-cell counts and a profile of dispensed antiretroviral therapy are routinely maintained, including the: prescription fill dates, medications prescribed, and amount dispensed. Records of dispensed antiretroviral medications, (including nucleoside analogs, PI and NNRTI) were used to determine the drugs dispensed and available to each subject during each month of follow-up. Subjects were considered unexposed until the first month that a PI was dispensed. The exposure at the time of the lipid measurement was calculated as the cumulative number of consecutive months of drug exposure up until and including the month in which a measurement was taken. As we were interested in estimating changes that occur in plasma lipoproteins and triglycerides within the first year after treatment initiation, we included measurements with a cumulative PI exposure up to a maximum of twelve months. Subjects were considered unexposed thirty days after the last PI was dispensed and, at that time, the cumulative exposure was set to zero. Plasma lipoproteins and triglycerides Approximately one-half of HIV-infected patients in BC are followed at one treatment centre located at St. Paul's Hospital, a large teaching hospital in Vancouver. For these subjects, in addition to virological testing, the hospital laboratory records the results of plasma lipoproteins and triglycerides. Subjects were routinely instructed to fast for 12 hours prior to the sample being drawn. Blood samples were collected in 10-1 EDTA-coated vacutainer tubes. Plasma was separated by centrifugation for 10 minutes at 2,000 revolutions per minute. TChol in the plasma was determined using an enzymatic method[ 14 ] and plasma triglyceride was determined as previously described[ 15 ]. HDL cholesterol was determined using a heparin manganese precipitation of apo B-containing lipoproteins[ 16 ] and LDL cholesterol was calculated using the Friedewald formula[ 17 , 18 ]. For some subjects, the first lipid measurement was taken prior to the dispensation of any antiretroviral drug ("treatment naïve" baseline lipid measurements). Other subjects had their first recorded plasma lipoproteins and triglycerides after HAART was initiated (initiation lipid levels). Statistical analysis We used linear mixed effects models[ 19 ] to estimate the effect of PI-based HAART on changes in plasma lipoproteins and triglycerides over twelve months. As blood lipid levels were always greater than 0, the responses were log transformed prior to model fitting. Effect estimates were obtained via restricted maximum likelihood estimation using the R © function lme(.). An analysis of variance indicated that simple parallel-line models, where the exposure effect was assumed to be constant over subjects, could adequately explain variation in the data compared to more complex models with random slopes. To account for correlated responses due to repeated measurements on the same individual, the correlation structure of the model was specified to include only a random intercept for each subject. The following explanatory variables were included in the models: age, sex, and CD4+ T- cell count at PI initiation, concomitant use of NNRTI, and a measure of adherence to antiretroviral therapy. Exposure to NNRTI was considered time-dependent and quantified using the same algorithm that was used to calculate time dependent PI exposure. Adherence was calculated by dividing the number of months of antiretroviral medications dispensed by the number of months of follow-up in the first twelve months after treatment initiation. Incomplete adherence represents the gap between the time that the previous medication supply ran out until the next refill date, and/or until the last contact date with the program. This method is reliably associated with both clinical outcomes and un-timed drug level monitoring[ 20 , 21 ]. For each outcome and associated regression coefficient β, the quantity exp(β)-1 represented the adjusted monthly percent change in that plasma lipid fraction. These values were annualized using the formula exp(12 × β)-1. Ninety five percent confidence intervals (95% CI) for the effect estimates were obtained using the standard error for each regression coefficient. Two sensitivity analyses were conducted. The first analysis involved restricting the analysis to lipid measurements which were taken on subjects prior to any change from baseline antiretroviral therapy. The second sensitivity analysis included only subjects who had lipid measurements taken prior to initiation of therapy. Results There were 679 subjects who were eligible for analysis, 91% of whom were male (Table 1 ). The median age was 38 years at initiation of therapy and the median baseline CD4+ T-CELL COUNT was 210 cells/mm 3 . All subjects initiated therapy that included a nucleoside analog and a PI. Table 1 Demographic and clinical characteristics at treatment program enrollement among 679 subjects treated with PI-based HAART initiation for HIV/AIDS at St Paul's Hospital, Vancouver, BC, August 1996 – January 2002 Characteristic* Age (y) Mean (SD) Median 39 (8.9) 38 Sex (% male) 91 Status (% alive) at end of follow-up 93 CD4 (cells/mm 3 ) Mean (SD) Median 255 (222) 210 Abbreviations: SD = Standard deviation; PI protease inhibitor. Subjects were followed for a median of 47 months (Table 2 ). During that follow-up time, subjects had a mean of 73% of months in which a PI had been dispensed, with a mean of 31 prescription refills. The mean adherence in the first year was 89%, and the median adherence was 100% indicating that more than one-half of subjects were dispensed all their medications. A total of 3,010 lipid measurements were used in the analysis, with 400 subjects having had two or more lipid measurements during the course of follow-up. There were a median of 3 (IQR 1–6) measurements per subject. The median time between lipid measurements was 3 (IQR 2–5) months for all subjects. Table 2 Use of PIs among 679 subjects initiating PI-based HAART for HIV/AIDS at St Paul's Hospital, Vancouver, BC, August 1996 – January 2002 Characteristic* Number of months between initiation of PI and end of observation, median (IQR)** 47 (29–62) % of follow-up time with dispensed therapy mean (SD), median for PI 73 (30) 84 Mean (SD) number prescriptions for*** PI 31 (19) % adherence in first year mean (SD) 89 (21) median (IQR) 100 (92 – 100) Abbreviations: SD = Standard deviation; IQR = Interquartile range; NRTI = nucleoside reverse transcriptase inhibitors; PI protease inhibitor; NNRTI = non-nucleoside reverse transcriptase inhibitor * PIs available during follow-up included: indinavir, nelfinavir, saquinavir, and ritonavir. All data taken from time of first therapy initiation (treatment program enrollement). ** Time between first dispensation of HAART (enrollment) and last date of follow-up. *** All prescriptions are of 30 day duration Table 3 shows the percentage change in plasma lipoprotein and triglyceride levels after 12 months of PI use, after adjustment for age, sex, and CD4+ T- cell count at initiation, concomitant use of NNRTI, and adherence. Statistically significant increases of about 20% were observed in TChol, the lipoprotein fractions, and triglycerides. Table 3 Baseline plasma cholesterol and triglycerides and estimated percent changes after 12 months when using PI among 679 subjects treated for HIV/AIDS at St Paul's Hospital, Vancouver, BC, August 1996 – January 2002 Number of measurements (subjects) Mean lipid baseline measurements (SD) % change* (95% CI) TChol 1620 (529) 4.2 (1.0) 20 (17, 24) HDL 1250 (419) 1.0 (0.3) 22 (15, 29) LDL 677 (295) 2.5 (0.8) 12 (5, 20) N-HDL 1247 (419) 3.1 (1.2) 20 (15, 26) TRG 1743 (556) 1.9 (1.2) 22 (12, 33) Abbreviations: PI protease inhibitor; TChol = total cholesterol; HDL = high-density lipoprotein cholesterol; LDL = low- density lipoprotein cholesterol; N-HDL = Non-HDL Cholesterol; TRG = triglycerides * Adjusted for age, sex, and CD4 + cell count at first prescription for HAART, concomitant use of non-nucleoside reverse transcriptase inhibitor and adherence to antiretroviral therapy in the first year of treatment The sensitivity analyses indicated that the models were robust to changes in the sub-population studied and the exposures that were included. In the analysis that included subjects who had a baseline measurement prior to start of HAART, a plasma profile prior to antiretroviral therapy initiation showed a similar pattern but with wider confidence intervals. In the analysis which was restricted to lipid measurements taken on subjects prior to switching from baseline therapy, the effect estimates were similar to those in Table 3 . Discussion In patients treated for HIV infection with HAART in a naturalistic setting, we observed that treatment with PIs was estimated to result in 20% increases in the levels of total cholesterol, HDL- and LDL-cholesterol and triglycerides twelve months after treatment initiation. Our results contribute to the growing body of evidence implicating PIs in the development of blood lipid abnormalities[ 22 ] and are in keeping with findings of randomized trials where a change from PI-based HAART to NRTI- or NNRTI-based HAART was associated with improvements in the lipid profile[ 23 ]. The observed increases in LDL-cholesterol and triglycerides were expected based on results reported from randomized trials. The finding of an increase in HDL-cholesterol has been reported only in some[ 24 ] but not all randomized trials. Low HDL levels and other forms of dyslipidemia, and disturbances in glucose metabolism, as well as central obesity, have been shown to be strong independent risk factors for cardiovascular morbidity and mortality in the general population[ 25 , 26 ]. Furthermore, the clustering of risk factors leads to greater cardiovascular morbidity and mortality than would be expected to occur in relation to each component alone[ 25 ]. These factors, when considered together, provide grounds to suspect that persons being treated for HIV infection with PI are at an increased risk of cardiovascular disease[ 8 ]. The increases in triglycerides caused by use of PI is of concern because there is growing evidence that these lipids are an independent risk factor for cardiovascular disease[ 27 , 28 ]. Two large observational cohort studies, published in 2003, showed discrepant results regarding the risks of cardiovascular disease among persons treated with HAART[ 29 , 30 ]. A retrospective administrative claims database study from the US Veteran's Affairs indicated that there was no relation between the use of antiretroviral therapy and the risk of cardiovascular or cerebrovascular hospitalizations [ 29 ]. A prospective multi-country collaborative study, with clinical events validation from eleven cohorts of HIV-infected persons showed that the incidence of myocardial infarction increased with longer exposure to combination antiretroviral therapy[ 30 ]. This study has a number of features that add credence to the results. First, while only based at one hospital, the study population comprised about one-half of the treated population in BC. The study sample had similar demographic composition (age and sex) and CD4+ T-cell count at initiation of therapy as other treated subjects in the province. We believe that these results are therefore generalizable to PI-regimen treated HIV-infected patients in BC, as well as to other target populations with similar demographic characteristics. Second, as all dispensed PIs and other antiretroviral medications are paid for and recorded centrally, there was complete information on all HAART medications available to all study subjects. We adjusted for adherence using each subjects' refill compliance in the first year of treatment. Third, data on the plasma lipid profile, including both lipoproteins and triglycerides, were of high quality. The measurements, while lacking the feature of being taken at uniformly spaced intervals as in closely monitored system such as randomized trial, represent the actual experience in an observational setting. Fourth, the enrollment period lasted over five years, allowing a large sample size. We employed repeated measures analyses to account for the correlated structure of the data. This study was also subject to several limitations. First, lipid measurements were not collected in a predetermined manner as is typical in a prospective research study. Consequently, the timing of measurements was irregular and not all lipid fractions were measured when blood was drawn. Second, despite instructions some subjects may not have fasted for the full 12 hour period prior to blood draws. Non-fasting triglyceride measurements are still useful for predicting cardiovascular disease and death[ 31 ]. Third, other potent risk factors for changes in lipid profiles such as family history and diet were not considered. It is possible but unlikely that the type of triple therapy prescribed by physicians could have been influenced by the belief that PIs may negatively impact lipid levels, likely leading to an attenuation of the estimates. Fourth, a number of factors contributed to a bias towards underestimating the true changes: for some subjects baseline values were not obtained prior to treatment initiation; it was not possible to adjust for concomitant use of lipid modifying agents such as statins because these data were not available; subjects with large changes in their lipid profiles may have been switched to another class; and treatment interruptions would also have affected exposure estimates. As a result of these limitations, the actual changes in plasma lipoprotein and triglycerides may be greater than reported in this study. Fifth, we assumed that patients who stop therapy or skip a month of therapy can be modelled as treatment naïve with respect to any subsequent therapy. More sophisticated statistical techniques are available[ 32 ] for handling this limitation, but appropriate software and resolution of technical issues are still in the developmental stages. Lastly, it is possible that the PI effect on lipids and triglyceride levels is not a class effect. The recently introduced PI, atazanavir sulfate, has been shown in several randomized clinical trials not to adversely affect lipid levels [ 33 - 38 ]. Due to changes in the HAART regimen that occurred because of resistance, non-compliance, or other reasons, it was not possible to isolate the effect of individual PIs. Over the period under study, most subjects initiating PI-based HAART were prescribed either indinavir or nelfinavir. Treating PI-induced dyslipidemia As PI-based HAART is very effective in increasing survival in HIV-infected patients[ 39 ], discontinuing PI is undesirable, even in patients with dyslipidemia. However, as PI-induced dyslipidemia is often asymptomatic and typically occurs in younger patients whose baseline risk of cardiovascular disease is low, the need for primary prevention is often under-appreciated in clinical practice[ 40 ]. When it is recognized, rather than discontinuing or switching PI therapy, one response is to initiate pharmacologic therapy with statins and/or fibrates[ 41 ]. Statins have been shown to be effective drugs to prevent cardiovascular disease in non-HIV patients[ 26 ]. While the role of statins in preventing cardiovascular disease in HIV patients remains to be demonstrated definitively, several smaller randomized controlled trials that have shown that these medications have beneficial effects on PI-induced dyslipidemia [ 42 ]. Until recently, pravastatin was the preferred statin for treating PI-induced dyslipidemia because it does not require metabolism by the cyp 3A4 system. However, recent studies have shown that moderate lipid lowering with pravastatin was less effective than intensive lipid lowering with atorvastatin in reducing: progression of atherosclerotic lesions [ 43 ] in patients requiring coronary angiography; and death or major cardiovascular events in patients with an acute coronary syndrome[ 44 ]. Atorvastatin is a powerful and effective statin, though there are still only limited published studies with this agent in HIV patients[ 45 ]. Because of metabolism by cyp 3A4, the dose of atorvastatin should be reduced one-half in patients receiving PIs. Lovastatin and simvastatin require metabolic activation by cyp 3A4 and should be avoided in patients receiving PIs. Rosusvastatin, a recently introduced statin has features that make it attractive for treating patients with PI-induced dyslipidemias: it does not require metabolism by the cyp 3A4 system and it effectively reduces LDL cholesterol. The safety and effectiveness of rosuvastatin in reducing clinical outcomes in HIV patients remain to be demonstrated. Fibrates are effective in lowering triglycerides and increasing HDL and have been shown to be effective in HIV patients[ 42 ]. Despite the impact on plasma lipids and triglycerides, only one trial has shown fibrates to reduce clinical outcomes[ 46 , 47 ]. Therefore, the role of fibrates in reducing cardiovascular disease outcomes remains to be fully elucidated. The main role of fibrates is to reduce the risk of pancreatitis associated with hypertriglyceridemia. In HIV patients, gemfibrozil should be limited to monotherapy because of the risk for myopathy. Fenofibrate is the preferred drug if combination therapy with a statin is contemplated. The efficacy of other lipid lowering drugs such as niacin and cholesterol transport blockers (ezetimibe) remains to be demonstrated. Bile acid sequestrants are contraindicated in patients receiving HAART. We conclude that there was an estimated 20% increase in lipoproteins and triglycerides in the first year after initiating PI-based antiretroviral therapy in HIV-infected patients under conditions of standard treatment. Whether these increases continue beyond one year is of considerable interest because lipoproteins and triglycerides are known risk factors for cardiovascular disease. In conjunction with the predominance or men, high rates of smoking, and aging of the treated HIV-positive population, elevated lipoproteins and triglycerides may mean that patients such as these are at elevated risk for cardiovascular events in the future. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549538.xml |
524515 | A step ahead: combining protein purification and correct folding selection | The success of recombinant protein expression seems unpredictable and even good yields of soluble proteins do not guarantee the correct folding. The search for soluble constructs can be performed by exploiting libraries and speeded up by automation, but these approaches are money and time consuming and the tags used for affinity purification can mask the real stability of the target proteins. The ideal purification protocol would include the structure quality control. A recent paper commented in this article describes a phage-display method to screen for antibodies that are able to re-fold after heat-denaturation and can be selectively affinity-purified only if monodispersed. It turned out that the proteins with high recovery performance after heat-shock were also suitable for efficient recombinant expression. | Introduction The possibility to produce recombinant proteins instead of recovering the native molecules offers the double advantage of higher yields and of a simplified purification protocol using affinity chromatography. At least half a dozen of the purification tags that have been proposed so far are routinely fused to the target proteins and used to perform affinity purification. E. coli is the most popular host for the expression of heterologous proteins but its simplified cell organization can be limiting for the expression of correctly folded recombinant proteins. No bioinformatic tools can predict if a construct will be expressed soluble in bacteria and, therefore, time-consuming cloning steps and expression optimization tests must be considered. In most of the cases the affinity purification protocols are effective. However, the costs of the resins and proteases necessary to remove the tags can become a limiting factor. Furthermore, the different requirements for the chromatography steps and proteolysis make difficult to conceive automatic systems for obtaining purified homogeneous protein. As an alternative, we showed that the fusion of a target protein with a thermostable partner can be purified to homogeneity by heating [ 1 ]. Because the recovered target proteins resulted correctly folded only in some cases the method seems rather suitable for the preparation of antigens than for functionally active molecules. We do not expect that heated proteins recover the native structural features but it is a common simplification to assume that a soluble protein is correctly folded and companies commercialize vectors with tags that "improve the solubility". Completely underestimated is the fact that a very soluble fusion partner can keep in solution unfolded target proteins. The work of the group around Travé showed the false results generated using fusion proteins and suggested a method for the evaluation of the aggregation state [ 2 , 3 ] and to consider the monodispersity as required parameter. Therefore, methods would be envisaged that combine the purification to the selection for the correct folding. An important contribution in this direction is the recent paper published by Greg Winter's group [ 4 ]. Discussion The authors [ 4 ] describe a method for selecting antibody heavy chain variable regions resistant to the heat-induced aggregation. The antibody domains were displayed at the tip of filamentous bacteriophage and recovered by affinity binding to Protein A or to the specific antigen after the heating step. The purification was dependent on the correct folding of the antibodies since aggregates did not bind to the ligand. Once expressed in bacteria the selected antibodies showed a high yield and the property of reversible unfolding. In conclusion, the selection for the feature "re-folding from denatured/aggregation state" enabled the isolation of constructs adapted to recombinant expression. The results suggest that for the selected proteins the mechanisms leading to the re-folding into the native state are common to those that organize the folding of linear amino acid chains. Functional genomic relies on the possibility of screening fast and efficiently large number of clones for their expression and correct folding. Several approaches have been suggested over the past years. An indirect method considers marker genes activated by misfolding [ 5 ] to discriminate aggregation-prone constructs. Otherwise, the solubility of reporter fusion partners has been considered [ 6 , 7 ]. Nevertheless, as well as for fusions with MBP or GST the correct folding of the fusion partner does not automatically mean that the target protein reached its native structure. The elegance of the method described by Winter and co-workers relies on the use of a ligand that recognizes only the folded state of the protein to purify: the quality control is inclusive in the affinity purification. Furthermore, the phage-display format allows for the identification of the corresponding clone. As pointed out by the authors, such an approach is limited to those cases for which a conformation-dependent bait is available or, at least, a reliable method exists to discriminate between native and aggregated proteins. The logic of the experiment reminds me to the protocol used to select in vivo , directly and exclusively, for the class of conventional antibodies able to fold in the cytoplasm [ 8 ]. It is difficult to envisage a method applicable to all protein classes for selecting constructs that will express correctly folded proteins. Nevertheless, it is still possible to improve the yield of recombinant proteins that tend to aggregate. At least part of the unfolded proteins is not definitely trapped in aggregates. Re-solubilisation from bacterial inclusion bodies happens in vivo [ 9 ], the involvement of specific chaperones in the disaggregation process has been illustrated [ 10 , 11 ] and we used their co-expression to boost the bacteria re-folding machinery and increase the recombinant target protein yield [ 12 ]. Our unpublished data show that the chaperone-dependent solubilised protein is correctly folded, namely the recombinant chaperones are integrated into the in vivo protein quality control. Conclusions The recombinant expression of proteins often induces the formation of soluble aggregates and several of such aggregates conserve sufficient features for being recovered by affinity chromatography. The simplification of having considered a purified soluble protein as a protein in native state has generated false results [ 2 ]. Therefore, methods that enable to selectively purify only correctly folded proteins [ 4 ] are welcome because couple purification and quality control. Unfortunately, their application is limited to few single protein classes for which a suitable binder exists. One important information of the Winter's group article is that the protein recovery after heat-shock, namely the possibility to re-fold correctly, correlates with correct folding in recombinant expression. Since the measurements of the aggregation index needs very small amounts of proteins [ 3 ] it would maybe worthy to screen using the aggregation index of heated domains to check if the Winter's group observation is a general rule and its application useful to select potentially soluble constructs in absence of specific binders. Finally, the heat-selection can provide useful insights about the molecular features involved in the (re)-folding/disaggregation mechanisms. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524515.xml |
393292 | Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones | The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/ ). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology. | Introduction The draft sequences of the human, mouse, and rat genomes are already available ( Lander et al. 2001 ; Marshall 2001 ; Venter et al. 2001 ; Waterston et al. 2002 ). The next challenge comes in the understanding of basic human molecular biology through interpretation of the human genome. To display biological data optimally we must first characterize the genome in terms of not only its structure but also function and diversity. It is of immediate interest to identify factors involved in the developmental process of organisms, non-protein-coding functional RNAs, the regulatory network of gene expression within tissues and its governance over states of health, and protein–gene and protein–protein interactions. In doing so, we must integrate this information in an easily accessible and intuitive format. The human genome may encode only 30,000 to 40,000 genes ( Lander et al. 2001 ; Venter et al. 2001 ), suggesting that complex interdependent gene regulation mechanisms exist to account for the complex gene networks that differentiate humans from lower-order organisms. In organisms with small genomes, it is relatively straightforward to use direct computational prediction based upon genomic sequence to identify most genes by their long open reading frames (ORFs). However, computational gene prediction from the genomic sequence of organisms with short exons and long introns can be somewhat error-prone ( Ashburner 2000 ; Reese et al. 2000 ; Lander et al. 2001 ). Previous efforts to catalogue the human transcriptome were based on expressed sequence tags (ESTs) used for the identification of new genes ( Adams et al. 1991 ; Auffray et al. 1995 ; Houlgatte et al. 1995 ), chromosomal assignment of genes ( Gieser and Swaroop 1992 ; Khan et al. 1992 ; Camargo et al. 2001 ), prediction of genes ( Nomura et al. 1994 ), and assessment of gene expression ( Okubo et al. 1992 ). Recently, Camargo et al. (2001 ) generated a large collection of ORF ESTs, and Saha et al. (2002 ) conducted a large-scale serial analysis of gene expression patterns to identify novel human genes. The availability of human full-length transcripts from many large-scale sequencing projects ( Nomura et al. 1994 ; Nagase et al. 2001 ; Wiemann et al. 2001 ; Yudate 2001; Kikuno et al. 2002 ; Strausberg et al. 2002 ) has provided a unique opportunity for the comprehensive evaluation of the human transcriptome through the annotation of a variety of RNA transcripts. Protein-coding and non-protein-coding sequences, alternative splicing (AS) variants, and sense–antisense RNA pairs could all be functionally identified. We thus designed an international collaborative project to establish an integrative annotation database of 41,118 human full-length cDNAs (FLcDNAs). These cDNAs were collected from six high-throughput sequencing projects and evaluated at the first international jamboree, entitled the Human Full-length cDNA Annotation Invitational (H-Invitational or H-Inv) ( Cyranoski 2002 ). This event was held in Tokyo, Japan, and took place from August 25 to September 3, 2002. Efforts which have been made in the same area as the H-Inv annotation work include the Functional Annotation of Mouse (FANTOM) project ( Kawai et al. 2001 ; Bono et al. 2002 ; Okazaki et al. 2002 ), Flybase (GOC 2001), and the RIKEN Arabidopsis full-length cDNA project ( Seki et al. 2002 ). In our own project, great effort has been taken at all levels, not only in the annotation of the cDNAs but also in the way the data can be viewed and queried. These aspects, along with the applications of our research to disease research, distinguish our project from other similar projects. This manuscript provides the first report by the H-Inv consortium, showing some of the discoveries made so far and introducing our new database of the human transcriptome. It is hoped that this will be the first in a long line of publications announcing discoveries made by the H-Inv consortium. Here we describe results from our integrative annotation in four major areas: mapping the transcriptome onto the human genome, functional annotation, polymorphism in the transcriptome, and evolution of the human transcriptome. We then introduce our new database of the human transcriptome, the H-Invitational Database (H-InvDB; http://www.h-invitational.jp ), which stores all annotation results by the consortium. Free and unrestricted access to the H-Inv annotation work is available through the database. Finally, we summarize our most important findings thus far in the H-Inv project in Concluding Remarks. Results/Discussion Mapping the Transcriptome onto the Human Genome Construction of the nonredundant human FLcDNA database We present the first experimentally validated nonredundant transcriptome of human FLcDNAs produced by six high-throughput cDNA sequencing projects ( Ota et al. 1997 , 2004 ; Strausberg et al. 1999 ; Hu et al. 2000 ; Wiemann et al. 2001 ; Yudate 2001 ; Kikuno et al. 2002 ) as of July 15, 2002. The dataset consists of 41,118 cDNAs (H-Inv cDNAs) that were derived from 184 diverse cell types and tissues (see Dataset S1 ). The number of clones, the number of libraries, major tissue origins, methods, and URLs of cDNA clones for each cDNA project are summarized in Table 1 . H-Inv cDNAs include 8,324 cDNAs recently identified by the Full-Length Long Japan (FLJ) project. The FLJ clones represent about half of the H-Inv cDNAs ( Table 1 ). The policies for library selection and the results of initial analysis of the constituent projects were reported by the participants themselves: the Chinese National Human Genome Center (CHGC) ( Hu et al. 2000 ), the Deutsches Krebsforschungszentrum (DKFZ/MIPS) ( Wiemann et al. 2001 ), the Institute of Medical Science at the University of Tokyo (IMSUT) ( Suzuki et al. 1997 ; Ota et al. 2004 ), the Kazusa cDNA sequence project of the Kazusa DNA Research Institute (KDRI) ( Hirosawa et al. 1999 ; Nagase et al. 1999 ; Suyama et al. 1999 ; Kikuno et al. 2002 ), the Helix Research Institute (HRI) ( Yudate et al. 2001 ), and the Mammalian Gene Collection (MGC) ( Strausberg et al. 1999 ; Moonen et al. 2002 ), as well as FLJ mentioned earlier ( Ota et al. 2004 ). The variation in tissue origins for library construction among these six groups resulted in rare occurrences of sequence redundancy among the collections. In a recent study, the FLJ project has described the complete sequencing and characterization of 21,243 human cDNAs ( Ota et al. 2004 ). On the other hand, the H-Inv project characterized cDNAs from this project and six high-throughput cDNA producers by using a different suite of computational analysis techniques and an alternative system of functional annotation. Table 1 Summary of cDNA Resources *FLcDNA data were provided for H-Inv project by the FLJ project of NEDO (URL: http://www.nedo.go.jp/bio-e/ ) and six high-throughput cDNA clone producers Chinese National Human Genome Center (CHGC), the Deutsches Krebsforschungszentrum (DKFZ/MIPS), Helix Research Institute (HRI), the Institute of Medical Science in the University of Tokyo (IMSUT), the Kazusa DNA Research Institute (KDRI), and the Mammalian Gene Collection (MGC/NIH) The 41,118 H-Inv cDNAs were mapped on to the human genome, and 40,140 were considered successfully aligned. The alignment criterion was that a cDNA was only aligned if it had both 95% identity and 90% length coverage against the genome ( Figure 1 ). The mean identity of all the alignments between 40,140 mapped cDNAs and genomic sequences was 99.6 %, and the mean coverage against the genomic sequence was 99.6%. In some cases, terminal exons were aligned with low identity or low coverage. For example, 89% of internal exons have identity of 99.8% or higher, while only 78% and 50% of the first and last exons do, respectively. These alignments with low identity or low coverage seemed to be caused by the unsuccessful alignments of the repetitive sequences found in UTR regions and the misalignments of 3′ terminal poly-A sequences. Although better alignments could be obtained for these sequences by improving the mapping procedure, we concluded that the quality of the FLcDNAs was high overall. Figure 1 Procedure for Mapping and Clustering the H-Inv cDNAs The cDNAs were mapped to the genome and clustered into loci. The remaining unmapped cDNAs were clustered based upon the grouping of significantly similar cDNAs. Due to redundancy and AS within the human transcriptome, these 40,140 cDNAs were clustered to 20,190 loci (H-Inv loci). For the remaining 978 unmapped cDNAs, we conducted cDNA-based clustering, which yielded 847 clusters. The clusters created had an average of 2.0 cDNAs per locus ( Table 2 ). The average was only 1.2 for unmapped clusters, probably because many of these genes are encoded by heterochromatic regions of the human genome and show limited levels of gene expression. The gene density for each chromosome varied from 0.6 to 19.0 genes/Mb, with an average of 6.5 genes/Mb. This distribution of genes over the genome is far from random. This biased gene localization concurs with the gene density on chromosomes found in similar previous reports ( Lander et al. 2001 ; Venter et al. 2001 ). This indicates that the sampled cDNAs are unbiased with respect to chromosomal location. Most cDNAs were mapped only at a single position on the human genome. However, 1,682 cDNAs could be mapped at multiple positions (with mean values of 98.2% identity and 98.1% coverage). The multiple matching may be caused by either recent gene duplication events or artificial duplication of the human genome caused by misassembled contigs. In our study we have selected only the “best” loci for the cDNAs (see Materials and Methods for details). Table 2 The Clustering Results of Human FLcDNAs onto the Human Genome a UN represents contigs that were not mapped onto any chromosome In total, 21,037 clusters (20,190 mapped and 847 unmapped) were identified and entered into the H-InvDB. We assigned H-Inv cluster IDs (e.g., HIX0000001) to the clusters and H-Inv cDNA IDs (e.g., HIT000000001) to all curated cDNAs. A representative sequence was selected from each cluster and used for further analyses and annotation. Comparison of the mapped H-Inv cDNAs with other annotated datasets In order to evaluate the H-Inv dataset, we compared all of the mapped H-Inv cDNAs with the Reference Sequence Collection (RefSeq) mRNA database ( Pruitt and Maglott 2001 ) ( Figure 2 ). The RefSeq mRNA database consists of two types of datasets. These are the curated mRNAs (accession prefix NM and NR) and the model mRNAs that are provided through automated processing of the genome annotation (accession prefix XM and XR). Figure 2 A Comparison of the Mapped H-Inv FLcDNAs and the RefSeq mRNAs The mapped H-Inv cDNAs, the RefSeq curated mRNAs (accession prefixes NM and NR), and the RefSeq model mRNAs (accession prefixes XM and XR) provided by the genome annotation process were clustered based on the genome position. The numbers of loci that were identified by clustering are shown. From the comparison, we found that 5,155 (26%) of the H-Inv loci had no counterparts and were unique to the H-Inv. All of these 5,155 loci are candidates for new human genes, although non-protein-coding RNAs (ncRNAs) (25%), hypothetical proteins with ORFs less than 150 amino acids (55%), and singletons (91%) were enriched in this category. In fact, 1,340 of these H-Inv-unique loci were questionable and require validation by further experiments because they consist of only single exons, and the 3′ termini of these loci align with genomic poly-A sequences. This feature suggests internal poly-A priming although some occurrences might be bona fide genes. The most reliable set of newly identified human genes in our dataset is composed of 1,054 protein-coding and 179 non-protein-coding genes that have multiple exons. Therefore, at least 6.1% (1,233/20,190) of the H-Inv loci could be used to newly validate loci that the RefSeq datasets do not presently cover. These genes are possibly less expressed since the proportion of singletons (H-Inv loci consisting of a single H-Inv cDNA) was high (84%). On the other hand, 78% (11,974/15,439) of the curated RefSeq mRNAs were covered by the H-Inv cDNAs. These figures suggest that further extensive sequencing of FLcDNA clones will be required in order to cover the entire human gene set. Nonetheless, this effort provides a systematic approach using the H-Inv cDNAs, even though a portion of the cDNAs have already been utilized in the RefSeq datasets. It is noteworthy that H-Inv cDNAs overlapped 3,061 (17%) of RefSeq model mRNAs, supporting this proportion of the hypothetical RefSeq sequences. These newly confirmed 3,061 loci have a mean number of exons greater than RefSeq model mRNAs that were not confirmed, but smaller than RefSeq curated mRNAs. The overlap between H-Inv cDNAs and RefSeq model mRNAs was smaller than that between H-Inv cDNAs and RefSeq curated mRNAs. This suggests that the genes predicted from genome annotation may tend to be less expressed than RefSeq curated genes, or that some may be artifacts. All these results highlight the great importance of comprehensive collections of analyzed FLcDNAs for validating gene prediction from genome sequences. This may be especially true for higher organisms such as humans. Incomplete parts of the human genome sequences The existence of 978 unmapped cDNAs (847 clusters) suggests that the human genome sequence (National Center for Biotechnolgy Information [NCBI] build 34 assembly) is not yet complete. The evidence supporting this statement is twofold. First, most of those unmapped cDNAs could be partially mapped to the human genome. Using BLAST, 906 of the unmapped cDNAs (corresponding to 786 clusters) showed at least one sequence match to the human genome with a bit score higher than 100. Second, most of the cDNAs could be mapped unambiguously to the mouse genome sequences. A total of 907 unmapped cDNAs (779 clusters; 92%) could be mapped to the mouse genome with coverage of 90% or higher. If we adopted less stringent requirements, more cDNAs could be mapped to the mouse genome. The rest might be less conserved genes, genes in unfinished sections of the mouse genome, or genes that were lost in the mouse genome. Based on these observations, we conclude that the human genome sequence is not yet complete, leaving some portions to be sequenced or reassembled. The proportion of the genome that is incomplete is estimated to be 3.7%–4.0%. The figure of 4.0% is based upon the proportion of H-Inv cDNA clusters that could not be mapped to the genome (847/21,037), while the 3.7% estimate is based on both H-Inv cDNAs and RefSeq sequences (only NMs). This statistic indicates that a minimum of one out of every 25–27 clusters appears to be unrepresented in the current human genome dataset, in its full form. Possible reasons for this include unsequenced regions on the human genome and regions where an error may have occurred during sequence assembly. If this is the case, this lends support to the use of cDNA mapping to facilitate the completion of whole genome sequences ( Kent and Haussler 2001 ). For example, we can predict the arrangement of contigs based on the order of mapped exons. In addition we can use the sequences of unmapped exons to search for those clones that contain unsequenced parts of the genome. The mapping results of partially mapped cDNAs are thus quite useful. Primary structure of genes on the human genome Using the H-Inv cDNAs, the precise structures of many human genes could be identified based on the results of our cDNA mapping ( Table S1 ). The median length of last exons (786 bp) was found to be longer than that of other exons, and the median length of first introns (3,152 bp) longer than that of other introns. These observed characteristics of human gene structures concur with the previous work using much smaller datasets ( Hawkins 1988 ; Maroni 1996 ; Kriventseva and Gelfand 1999 ). In the human genome, 50% of the sequence is occupied by repetitive elements ( Lander et al. 2001 ). Repetitive elements were previously regarded by many as simply “junk” DNA. However, the contribution of these repetitive stretches to genome evolution has been suggested in recent works ( Makalowski 2000 ; Deininger and Batzer 2002 ; Sorek et al. 2002 ; Lorenc and Makalowski 2003 ). The 21,037 loci of representative cDNAs were searched for repetitive elements using the RepeatMasker program. RepeatMasker indicated that 9,818 (47%) of the H-Inv cDNAs, including 5,442 coding hypothetical proteins, contained repetitive sequences. The existence of Alu repeats in 5% of human cDNAs was reported previously ( Yulug et al. 1995 ). Our results revealed a significant number of repetitive sequences including Alu in the human transcriptome. Among them, 1,866 cDNAs overlapped repetitive sequences in their ORFs. Moreover, 554 of 1,866 cDNAs had repetitive sequences contained completely within their ORFs, including 81 cDNAs that were identical or similar to known proteins. This may indicate the involvement of repetitive elements in human transcriptome evolution, as suggested by the presence of Alu repeats in AS exons ( Sorek et al. 2002 ) and the contribution to protein variability by repetitive elements in protein-coding regions ( Makalowski 2000 ). We detected 2,254 and 5,427 cDNAs containing repetitive sequences in their 5′ UTR and 3′ UTR, respectively. The positioning of the repetitive elements suggests they play a regulatory role in the control of gene expression ( Deininger and Batzer 2002 ) (see Table S1 or the H-InvDB for details). AS transcripts We wished to investigate the extent to which the functional diversity of the human proteome is affected by AS. In order to do this, we searched for potential AS isoforms in 7,874 loci that were supported by at least two H-Inv cDNAs. We examined whether or not these cDNAs represented mutually exclusive AS isoforms, using a combination of computational methods and human curation (see Materials and Methods ). All AS isoforms that were supported independently by both methods were defined as the H-Inv AS dataset. Our analysis showed that 3,181 loci (40 % of the 7,874 loci) encoded 8,553 AS isoforms expressing a total of 18,612 AS exons. On average, 2.7 AS isoforms per locus were identified in these AS-containing loci. This figure represents half of the AS isoforms predicted by another group ( Lander et al. 2001 ). Our result highlights the degree to which full-length sequencing of redundant clones is necessary when characterizing the complete human transcriptome. The relative positions of AS exons on the loci varied: 4,383 isoforms comprising 1,538 loci were 5′ terminal AS variants; 5,678 isoforms comprising 1,979 loci were internal AS variants; and 2,524 isoforms comprising 921 loci were 3′ terminal AS variants. The AS isoforms found in the H-Inv AS dataset have strikingly diverse functions. Motifs are found over a wide range of protein sequences. For certain types of subcellular targeting signals, such as signal peptides, position within the entire protein sequence appears crucial. A total of 3,020 (35 %) AS isoforms contained AS exons that overlapped protein-coding sequences. 1,660 out of 3,020 AS isoforms (55%) harbored AS exons that encoded functional motifs. Additionally, 1,475 loci encoded AS isoforms that had different subcellular localization signals, and 680 loci had AS isoforms that had different transmembrane domains. These results suggest marked functional differentiation between the varying isoforms. If this is the case, it would appear that AS contributes significantly to the functional diversity of the human proteome. As the coverage of the human transcriptome by H-Inv cDNAs is incomplete, it would be misleading to conjecture that our dataset comprehensively includes all AS transcripts from every human gene. However, the current collection is a robust characterization of the existing functional diversity of the human proteome, and it represents a valuable resource of full-length clones for the characterization of experimentally determined AS isoforms. In the cases where three-dimensional (3D) structures could be assigned to H-Inv cDNA protein products, we have examined the possible impact of AS rearrangements on the 3D structure. Our analysis was performed using the Genomes TO Protein structures and functions database (GTOP) ( Kawabata et al. 2002 ). We found that some of the sequence regions in which internal exons vary between different isoforms contained regions encoding SCOP domains ( Lo Conte et al. 2000 ). This discovery allowed us to perform a simple analysis of the structural effects of AS. Our analysis of the SCOP domain assignments revealed that the loci displaying AS are much more likely to contain class c (β–α–β units, α/β) SCOP domains than class d (segregated α and β regions, α+β) or class g (small) domains. An example of exon differences between AS isoforms is presented in Figure 3 . The structures shown are those of proteins in the Brookhaven Protein Data Bank (PDB) ( Berman et al. 2000 ) to which the amino acid sequences of the corresponding AS isoforms are aligned. Segments of the AS isoform sequences that are not aligned with the corresponding 3D structure are shown in purple. Figure 3 demonstrates that exon differences resulting from AS sometimes give rise to significant alternations in 3D structure. Figure 3 An Example of Different Structures Encoded by AS Variants Exons are presented from the 5′ end, with those shared by AS variants aligned vertically. The AS variants, with accession numbers AK095301 and BC007828, are aligned to the SCOP domain d.136.1.1 and corresponding PDB structure 1byr. Helices and beta sheets are red and yellow, respectively. Green bars indicate regions aligned to the PDB structure, while open rectangles represent gaps in the alignments. AK095301 is aligned to the entire PDB structure shown, while BC007828 is lacking the alignment to the purple segment of the structure. Functional Annotation We predicted the ORFs of 41,118 H-Inv cDNA sequences using a computational approach (see Figure S1 ), of which 39,091 (95.1%) were protein coding and the remaining 2,027 (4.9%) were non-protein-coding. Since the structures and functions of protein products from AS isoforms are expected to be basically similar, we selected a “representative transcript” from each of the loci (see Figure S2 ). Then we identified 19,660 protein-coding and 1,377 non-protein-coding loci ( Table 3 ). Human curation suggested that a total of 86 protein-coding transcripts should be deemed questionable transcripts. Once identified as dubious these sequences were excluded from further analysis. The remaining representatives from the 19,574 protein-coding loci were used to define a set of human proteins (H-Inv proteins). The tentative functions of the H-Inv proteins were predicted by computational methods. Following computational predictions was human curation. Table 3 Statistics Obtained from the Functional Annotation Results After determination of the H-Inv proteins, we performed a standardized functional annotation as illustrated in Figure 4 , during which we assigned the most suitable data source ID to each H-Inv protein based on the results of similarity search and InterProScan. We classified the 19,574 H-Inv proteins according to the levels of the sequence similarity. Using a system developed for the human cDNA annotation (see Figure S2 ), we classified the H-Inv proteins into five categories ( Table 3 ). Three categories contain translated gene products that are related to known proteins: 5,074 (25.9%) were defined as identical to a known human protein (Category I proteins); 4,104 (21.0%) were defined as similar to a known protein (Category II proteins); and 2,531 (12.9%) as domain-containing proteins (Category III proteins). In total, we were able to assign biological function to 59.9% of H-Inv proteins by similarity or motif searches. The remaining proteins, for which no biological functional was inferred, were annotated as conserved hypothetical proteins (Category IV proteins; 1,706, 8.7%) if they had a high level of similarity to other hypothetical proteins in other species, or as hypothetical proteins (Category V proteins; 6,159, 31.5%) if they did not. Figure 4 Schematic Diagram of Human Curation for H-Inv Proteins The diagram illustrates the human curation pipeline to classify H-Inv proteins into five similarity categories; Category I , II, III, IV, and V proteins. To predict the functions of hypothetical proteins (Category IV and V proteins), we used 196 sequence patterns of functional importance derived from tertiary structures of protein modules, termed 3D keynotes ( Go 1983 ; Noguti et al. 1993 ). Application of the 3D keynotes to the H-Inv proteins resulted in the prediction of functions in 350 hypothetical proteins (see Protocol S1 ). Features of ORFs deduced from human FLcDNAs The mean and median lengths of predicted ORFs were calculated for the 19,574 H-Inv proteins. These were 1,095 bp and 806 bp, respectively ( Table 4 ). The values obtained were smaller than those from other eukaryotes, and are inconsistent with estimates reported previously ( Shoemaker et al. 2001 ). However, as has been seen in the earlier annotation of the fission yeast genome ( Das et al. 1997 ), our dataset might contain stretches which mimic short ORFs. This would lead to a bias in our ORF prediction and result in an erroneous estimate of the average ORF length. We examined the size distributions of ORFs from the five categories, and found that the distribution pattern was quite similar across categories. The exception was Category V, in which short ORFs were unusually abundant ( Figure S3 ). Judging from the length distribution of ORFs in the five categories of H-Inv proteins, the majority of ORFs shorter than 600 bps in Category V seemed questionable. In order to have a protein dataset that contains as many sequences to be further analyzed as possible, we have taken the longest ORFs over 80 amino acids if no significant candidates were detected by the sequence similarity and gene prediction (see Figure S1 ). The consequence of this is that Category V appears to contain short questionable ORFs, a certain fraction of which may be prediction errors. Nevertheless, these ORFs could be true. It is also possible that those ORFs were in fact translated in vivo when we curated the cDNAs manually. The existence of many functional short proteins in the human proteome is already confirmed, and there are 199 known human proteins that are 80 amino acids or shorter in the current Swiss-Prot database. We think that the H-Inv hypothetical proteins require experimentally verification in the future. Excluding the hypothetical proteins from the analysis, we obtained mean and median lengths for the ORFs of 1,368 bp and 1,130 bp, respectively, which are reasonably close to those for other eukaryotes ( Table 4 ). Table 4 The Features of Predicted ORFs Nonredundant proteome datasets of nonhuman species were obtained from the following URLs: fly ( Drosophila melanogaster ; http://flybase.bio.indiana.edu/ ), worm ( Caenorhabditis elegans ; http://www.wormbase.org/ ), budding yeast ( Saccharomyces cerevisiae ; http://www.pasteur.fr/externe ), fission yeast ( Schizosaccharomyces pombe ; http://www.sanger.ac.uk/ ), plant ( Arabidopsis thaliana ; http://mips.gsf.de/proj/thal/index.html ), and bacteria ( Escherichia coli K12; http://www.ncbi.nlm.nih.gov/ ) Of the 4,104 Category II proteins, 3,948 proteins (96.2%) were similar to the functionally identified proteins of mammals ( Figure S4 ). This implies that the predicted functions in this study were based on the comparative study with closely related species, so that the functional assignment retains a high level of accuracy if we suppose that protein function is more highly conserved in more closely related species. Moreover, the patterns of codon usage and the codon adaptation index (CAI; http://biobase.dk/embossdocs/cai.html ) of H-Inv proteins were investigated ( Table S2 ). The results indicated that the ORF prediction scheme worked equally well in the five similarity categories of H-Inv proteins. Each H-Inv protein in the five categories was investigated in relation to the tissue library of origin ( Table S3 ). We found that at least 30% of the clones mainly isolated from dermal connective, muscle, heart, lung, kidney, or bladder tissues could be classified as Category I proteins. Hypothetical proteins (Category V), on the other hand, were abundant in both endocrine and exocrine tissues. This bias may indicate that expression in some tissues may not have been studied in enough detail. If this is the case, then there is likely a significant gap between our current knowledge of the human proteome and its true dimensions. Non-protein-coding genes Over recent years, ncRNAs have been found to play key roles in a variety of biological processes in addition to their well-known function in protein synthesis ( Moore and Steitz 2002 ; Storz 2002 ). Analysis of the H-Inv cDNA dataset revealed that 6.5% of the transcripts are possibly non-protein-coding, although the number is much smaller than that estimated in mice ( Okazaki et al. 2002 ). We believe that this difference between the two species is mainly due to the larger number of mouse libraries that were used and to a rare-transcript enrichment step that was applied to these collections. To identify ncRNAs, we manually annotated 1,377 representative non-protein-coding transcripts, which were classified into four categories (see Table 3 ; Figure 5 ): putative ncRNAs, uncharacterized transcripts (possible 3′ UTR fragments supported by ESTs), unclassifiable transcripts (possible genomic fragments), and hold transcripts (not stringently mapped onto the human genome). Of these, 296 (19.5%) were putative ncRNAs with no neighboring transcripts in the close vicinity (> 5 kb) and supported by ESTs with a poly-A signal or a poly-A tail, indicating that these may represent genuine ncRNA genes. On the other hand, a large fraction of the non-protein-coding transcripts (675; 44.5%) were classified as possible 3′ UTRs of genes that were mapped less than 5 kb upstream. The 5-kb range is an arbitrary distance that we defined as one of our selection criteria for identifying ncRNAs. However, authentic non-protein-coding genes might be located adjacent to other protein-coding genes (as described earlier). Thus, some of the transcripts initially annotated as uncharacterized ESTs may correspond to ncRNAs when these sequences satisfy the other selection criteria. Figure 5 The Manual Annotation Flow Chart of ncRNAs Candidate non-protein-coding genes were compared with the human genome, ESTs, cDNA 3′-end features and the locus genomic environment. The candidates were then classified into four categories: hold (cDNAs improperly mapped onto the human genome); uncharacterized transcripts (transcripts overlapping a sense gene or located within 5 kb of a neighboring gene with EST support); putative ncRNAs (multiexon or single exon transcripts supported by ESTs or 3′-end features); and unclassifiable (possible genomic fragments). We defined a manual annotation strategy ( Figure 5 ) that allowed us to select convincing putative ncRNAs with various lines of supporting evidence. These are the following: absence of a neighboring gene in the close vicinity, overlap with human or mouse ESTs, occurrence in the 3′ end of cDNA sequences, as well as overlap with mouse cDNAs. Out of 296 annotated putative ncRNAs, we identified 47 ncRNAs with conserved RNA secondary structure motifs ( Rivas and Eddy 2001 ), and nearly 60% of these were found expressed in up to eight human tissues (data not shown), indicating that the manual curation strategy employed in this study may facilitate the identification of novel non-protein-coding genes in other species. The functions of human proteins identified through an analysis of domains Proteins in many cases are composed of distinct domains each of which corresponds to a specific function. The identification and classification of functional domains are necessary to obtain an overview of the whole human proteome. In particular, the analysis of functional domains allows us to elucidate the evolution of the novel domain architectures of genes that life forms have acquired in conjunction with environmental changes. The human proteome deduced from the H-Inv cDNAs was subjected to InterProScan, which assigned functional motifs from the PROSITE, PRINTS, SMART, Pfam, and ProDom databases ( Mulder et al. 2003 ). A total of 19,574 H-Inv proteins were examined, and 9,802 of them (50.1%) were assigned at least one InterPro code that was classified into either repeats (a region that is not expected to fold into a globular domain on its own), domains (an independent structural unit that can be found alone or in conjunction with other domains or repeats), and/or families (a group of evolutionarily related proteins that share one or more domains/repeats in common) when compared with those of fly, worm, budding and fission yeasts, Arabidopsis thaliana, and Escherichia coli ( Table S4 ). Moreover, the proteins were classified according to the Gene Ontology (GO) codes that were assigned to InterPro entries ( Table S5 ). Identification of human enzymes and metabolic pathways One of the most important goals of the functional annotation of human cDNAs is to predict and discover new, previously uncharacterized enzymes. In addition, revealing their positions in the metabolic pathways helps us understand the underlying biochemical and physiological roles of these enzymes in the cells. We thus searched for potential enzymes among the H-Inv proteins, and mapped them to a database of known metabolic pathways. We could assign 656 kinds of potential Enzyme Commission (EC) numbers to 1,892 of the 19,574 H-Inv proteins based on matches to the InterPro entries and GO assignments and on the similarity to well-characterized Swiss-Prot proteins (see Dataset S2 ). The number of characterized human enzymes significantly increased through this analysis. The most abundant enzymes in the H-Inv proteins were protein–tyrosine kinases (EC 2.7.1.112), which is consistent with the large number of kinases found in the InterPro assignments. The other major enzymes were small monomeric GTPase (EC 3.6.1.47), adenosinetriphosphatase (EC 3.6.1.3), phosphoprotein phosphatase (EC 3.1.3.16), ubiquitin thiolesterase (EC 3.1.2.15), and ubiquitin-protein ligase (EC 6.3.2.19). These enzymes are members of large multigene families that are important for the functions of higher organisms. Furthermore, we could assign 726 EC numbers to mouse representative transcripts and proteins ( Okazaki et al. 2002 ), and most of them appeared to be shared between human and mouse (data not shown). The high similarity of the enzyme repertoire between these two species is not surprising if we consider the close evolutionary relatedness between them. It does, however, indicate the usefulness of the mouse as a model organism for studies concerning metabolism. We then mapped all H-Inv proteins on the metabolic pathways of the KEGG database, a large collection of information on enzyme reactions ( Kanehisa et al. 2002 ). In total, we mapped 963 H-Inv proteins on a total of 1,613 KEGG pathways, of which 641 were based on their EC number assignments ( Figure S5 ). Those based on EC number assignments do not necessarily function as they are assigned because they have yet to be verified experimentally. However, if all other enzymes along the same pathway exist in humans, the functional assignment has a high probability of being correct. Using this method, we discovered a total of 32 newly assigned human enzymes from the H-Inv proteins with the support of KEGG pathways ( Table S6 ). For example, we identified (1) pyridoxamine-phosphate oxidase (EC 1.4.3.5; AK001397), an enzyme in the “salvage pathway,” the function of which is the reutilization of the coenzyme pyridoxal-5′-phosphate (its role in epileptogenesis was recently reported [ Bahn et al. 2002 ]), (2) ATP-hydrolysing 5-oxoprolinase (EC 3.5.2.9; AL096750) that cleaves 5-oxo-L-proline to form L-glutamate (whose deficiency is described in the Online Mendelian Inheritance in Man [OMIM] database [ID=260005]), and (3) N-acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25; BC018734), which catalyzes N-acetylglucosamine at the second step of its catabolism, the activity of which in human erythrocytes was detected by a biochemical study ( Weidanz et al. 1996 ). Many of the newly identified enzymes were supported by currently available experimental and genomic data. An example is a putative urocanase (EC 4.2.1.49; AK055862) that mapped onto the “histidine metabolism” that urocanic acid catabolises. A 14 C Histidine tracer study unexpectedly revealed that NEUT2 mice deficient in 10-formyltetrahydrofolate dehydrogenase (FTHFD) excrete urocanic acid in the urine and lack urocanase activity in their hepatic cytosol ( Cook 2001 ). We then found that both the FTHFD and AK055862 genes were located within the same NCBI human contig (NT005588) on Chromosome 3. Moreover, the distance between the two genes was consistent with the genetic deletion of NEUT2 (> 30 kb). We thus assumed that FTHFD and urocanase might be coincidentally defective in mice. This analysis could confirm that the AK055862 protein is a true urocanase. This example demonstrates that this kind of in silico analysis is a powerful method in defining the functions of proteins. Polymorphism in the Transcriptome Sites of potential polymorphism in cDNAs Due to the rapidly increasing accumulation of genetic polymorphism data, it is necessary to classify the polymorphism data with respect to gene structure in order to elucidate potential biological effects ( Gaudieri et al. 2000 ; Sachidanandam et al. 2001 ; Akey et al. 2002 ; Bamshad and Wooding 2003 ). For this purpose, we examined the relationship between publicly available polymorphism data and the structure of our H-Inv cDNA sequences. A total of 4 million single nucleotide polymorphisms (SNPs) and insertion/deletion length variations (indels) with mapping information from the Single Nucleotide Polymorphism Database (dbSNP; http://www.ncbi.nlm.nih.gov/SNP/ , build 117) ( Sherry et al. 1999 ) were used for the search. We could identify 72,027 uniquely mapped SNPs and indels in the representative H-Inv cDNAs and observed an average SNP density of 1/689 bp. To classify SNPs and indels with respect to gene structure, the genomic coordinates of SNPs were converted into the corresponding nucleotide positions within the mapped cDNAs. The SNPs and indels were classified into three categories according to their positions: 5′ UTR, ORF, and 3′ UTR ( Table 5 ). The density of indels was higher in 5′ UTRs (1/15,999 bp) and 3′ UTRs (1/12,553 bp) than in ORFs (1/45,490 bp). This is possibly due to different levels of functional constraints. We also examined the length of indels and found a higher frequency of indels in those ORFs that had a length divisible by three and that did not change their reading frames. We observed that the density of SNPs was higher in both the 5′ and 3′ UTRs (1/569 bp and 1/536 bp, respectively) than in ORFs (1/833 bp). Table 5 The Numbers of SNPs and indels Occurring in the Representative cDNAs a The numbers of SNPs and indels are summarized for representative cDNA sequences which were mapped on the genome. The numbers in parentheses represent the densities of SNPs and indels b SNPs that cause nonsense mutation or extension of polypeptides were classified assuming that the cDNAs represent original alleles c This figure includes 64 unclassifiable SNPs SNPs located in ORFs were classified as either synonymous, nonsynonymous, or nonsense substitutions ( Table 5 ). We identified 13,215 nonsynonymous SNPs that affect the amino acid sequence of a gene product. At least 4,998 of these nonsynonymous SNPs are “validated” SNPs (as defined by dbSNP). This data can be used to predict SNPs that affect gene function. SNPs that create stop codons can cause polymorphisms that may critically alter gene function. We identified 358 SNPs that caused either a nonsense mutation or an extension of the polypeptide. We classified these 358 SNPs into these two types based on the alleles of the cDNA. Most of these SNPs (315/358) were predicted to cause truncation of the gene products and produce a shorter polypeptide compared with the alleles of H-Inv cDNAs. For example, Reissner's fiber glycoprotein I (AK093431) contains a nonsense SNP that results in the loss of the last 277 amino acids of the protein, and consequently the loss of a thrombospondin type I domain located in its C-terminal end. This SNP is highly polymorphic in the Japanese population, the frequencies of G (normal) and T (termination) being 0.43 and 0.57, respectively. As seen in this example, the identification of SNPs within cDNAs provides important insights into the potential diversity of the human transcriptome. Thus, polymorphism data crossreferenced to a comprehensively annotated human transcriptome might prove to be a valuable tool in the hands of researchers investigating genetic diseases. Sites of microsatellite repeats Among the 19,442 representative protein-coding cDNAs, we identified a total of 2,934 di-, tri-, tetra-, and penta-nucleotide microsatellite repeat motifs ( Table 6 ). Interestingly, 1,090 (37.2%) of these were found in coding regions, the majority of which (86.9%) were tri-nucleotide repeats. Di-, tetra-, and penta-nucleotide repeats made up the greatest proportion of repeats in 5′ UTRs and 3′ UTRs. Coding regions contained mostly tri-nucleotide repeats. This result is consistent with the idea that microsatellites are prone to mutations that cause changes in numbers of repeats. Only tri-nucleotide repeats can conserve original reading frames when extended or shortened by mutations. A previous study showed that many of the microsatellite motifs identified in human genomic sequences, including those in coding regions, are highly polymorphic in human populations ( Matsuzaka et al. 2001 ). We found this to be the case in our study: 36 of the microsatellite repeats we detected were found to be polymorphic in human populations according to dbSNP records (data not shown). We identified 216 microsatellite repeats in 213 genes that showed contradictory numbers of repeats between cDNA and genome sequences (see Dataset S3 ). This figure includes 25 microsatellites in ORFs that have the potential to alter the protein sequences. Individual cases need to be verified by further experimental studies, but many of these microsatellites may really be polymorphic in human populations and have marked phenotypic effects. Table 6 The Numbers of Microsatellite Repeat Motifs That Occurred in the Representative cDNAs Microsatellites were defined as those sequences having at least ten repeats for di-nucleotide repeats and at least five repeats for tri-, tetra-, and penta-nucleotide repeats. Numbers of polymorphic microsatellites inferred by comparisons of cDNA and genomic sequences are shown in parenthesis. See Table S2 for a list of accession numbers for these cDNAs There were 382 cDNAs that possessed two or more microsatellites in their nucleotide sequences. This is illustrated in RBMS1 (BC018951), a cDNA which encodes an RNA-binding motif. This cDNA has four microsatellites, (GGA) 7 , (GAG) 9 , (GAG) 6 , and (GCC) 6 , in its 5′ UTR. These microsatellites are all located at least 98 bp upstream of the start codon, but they could still have pronounced regulatory effects on gene expression. Another example is the cDNA that encodes CAGH3 (AB058719). This cDNA has four microsatellites, (CAG) 8 , (CAG) 6 , (CAG) 8 , and (CAG) 8, all of which are located within the ORF. These microsatellites all encode stretches of poly-glutamine, which are known to have transcription factor activity ( Gerber et al. 1994 ) and often cause neurodegenerative diseases when the number of repeats exceeds a certain limit. A typical example of a disorder caused by these repeats is Huntington's disease ( Andrew et al. 1993 ; Duyao et al. 1993 ; Snell et al. 1993 ). We also searched for repeat motifs containing the same amino acid residue in the encoded protein sequences. We located a total of 3,869 separate positions where the same amino acid was repeated at least five times. The most frequent repetitive amino acids are glutamic acid, proline, serine, alanine, leucine, and glycine. The glutamine repeats of this nature were found in 160 different locations. Evolution of the Human Transcriptome Beyond the study of individual genes, the comparison of numerous complete genome sequences facilitates the elucidation of evolutionary processes of whole gene sets. Moreover, the FLcDNA datasets of humans and mice give us an opportunity to investigate the genome-wide evolution of these two mammals by using the sequences supported by physical clones. Here we compared our human cDNA sequences with all proteins available in the public databases. Focusing on our results, we discuss when and how the human proteome may have been established during evolution. Furthermore, the evolution of UTRs is examined through comparisons with cDNAs from both primates and rodents. Conserved and derived protein-coding genes in humans An advantage of large-scale cDNA sequencing is that it can generate a nearly complete gene set with good evidence for transcription. The human proteome deduced from the FLcDNA sequences gives us an opportunity to decipher the evolution of the entire proteome. Here we compare the representative H-Inv cDNAs with the Swiss-Prot and TrEMBL protein databases using FASTY ( Pearson 2000 ), and we describe the distributions of the homologs among taxonomic groups at two different similarity levels. The number of representative H-Inv cDNAs that have homolog(s) in a given taxon was counted ( Figure S6 ), and the cDNAs were classified into functional categories ( Figure 6 ). These results indicated that homologs of the human proteins were probably conserved much more in the animal kingdom than in the others at both moderate ( E <10 −10 ) and weak ( E < 10 −5 ) similarity levels (see Figure S6 ). Moreover, human sequences had as many nonmammalian animal homologs as mammalian homologs, with seemingly little bias to any one function (see Figure 6 ). This suggests that the genetic background of humans may have already been established in an early stage of animal evolution and that many parts of the whole genetic system have probably been stable throughout animal evolution despite the seemingly drastic morphological differences between various animal species. This result is consistent with our previous observation that the distribution of the functional domains is highly conserved among animal species (see Table S4 ). The number of homologs may have been inflated by recent gene duplication events within the human lineage. Hence we counted the number of paralog clusters instead of cDNAs that had homologs in the databases, and obtained essentially the same results ( Figure S7 ). Figure 6 The Functional Classification of H-Inv Proteins That Are Homologous to Proteins in Each Taxonomic Group The numbers of representative H-Inv cDNAs with sequence homology to other species' proteins ( E < 10 −5 ) were calculated. The cDNAs for which we could not assign any functions were discarded. Mammalian species were excluded from the “animal” group. “Eukaryote” represents eukaryotic species other than those included in the mammal, animal, fungi, and plant groups. See also Table S7. This analysis also revealed a number of potential human-specific proteins, which did not have any homologs in the current sequence databases. In this case the creation of lineage-specific genes through speciation is not completely excluded. However, most ORFs with no similarity to known proteins would not be genuine for the reasons discussed above. Therefore, the number of “true” human-specific proteins is expected to be relatively small. We conducted further BLASTP searches matching entries from the Swiss-Prot database against the H-Inv dataset itself. As a result, 12,813 (45.3%) of 28,263 vertebrate proteins had homologs in nonvertebrates at E < 10 –30 . Taking into account that the dataset is relatively small (approximately 12,000 sequences) and as a result may be biased, animal species may conceivably share a similar protein-coding gene set. Ohno (1996 ) proposed that the emergence of a large number of animal phyla in a short period of time would endow them with almost identical genomes. These were collectively referred to as the pananimalia genome. Our data support Ohno's hypothesis from the perspective that the basic gene repertoires of animals are essentially highly similar among diverse species that have evolved separately since the Cambrian explosion. Subsequently, morphological evolution seems to have been brought about mainly by changes in gene regulation. The number of transcription regulator homologs is different between animals and other phyla ( Table S7 ). In this analysis it was not possible to examine the genes recently deleted from the human lineage. However, the similarity of the proteome sets between distantly related mammals such as human and mouse ( Waterston et al. 2002 ) suggests that not many genes have been deleted specifically from humans since humans and mice diverged. A unique feature of the Animalia proteome is, for example, the presence of apoptosis regulator homologs, which are found widely in the animal kingdom, whilst they are rare in the other phyla ( Table S7 ). Since apoptosis plays an important role during the development of multicellular animals, this observation indicates that apoptosis was established independently of both plants and fungi during the early evolution of multicellularization in the kingdom Animalia. Likewise, signal transducers and cell-adhesion proteins are distinctive. In contrast, enzymes, translation regulators, molecular chaperones, etc. were highly conserved among all taxonomic groups. These proteins may have played such essential roles that any alterations were eliminated by strong purifying selection. It is assumed some functions were presumably derived from ancient endocellular symbionts (mitochondria and chloroplasts) ( Martin 2002 ). Evolution of untranslated regions The UTRs of mRNA are known to be involved in the regulation of gene expression at the posttranscriptional level through control of translation efficiency ( Kozak 1989 ; Geballe and Morris 1994 ; Sonenberg 1994 ), mRNA stability ( Zaidi and Malter 1994 ; McCarthy and Kollmus 1995 ), and mRNA localization ( Curtis et al. 1995 ; Lithgow et al. 1997 ). Only a few studies on very limited datasets have been carried out so far to describe quantitatively either the evolutionary dynamics of mRNA UTRs ( Larizza et al. 2002 ), or their general structural and compositional features ( Pesole et al. 1997 ). The human transcriptome presented here along with the murid data obtained mainly from the FANTOM2 project enables us to stabilize a mammalian genome perspective on the subject ( Table S8 ). A sliding window analysis of UTR sequence identities between humans and mice revealed a positive correlation between the number of indels in an untranslated region and the distance from the coding sequence ( Figure 7 ). Unlike indels, mismatches are distributed equally along whole untranslated regions. In other words, indels seem to be less tolerated in close proximity to a coding sequence, while substitutions are evenly distributed along the untranslated regions of the mRNAs. This seems to be a general pattern observed similarly in other species (data not shown). Indels in UTRs may have been avoided so that the distance between the coding region and a signal sequence for regulation in the UTR could be conserved throughout evolution, while purifying selection against substitutions appeared to be relatively weak. Figure 7 Window Analysis of Similarity between Human and Mouse UTRs Results for 5′ UTRs presented above and for 3′ UTRs below. The whole mRNA sequences were aligned using a semiglobal algorithm as implemented in the map program (Huang 1994) with the following parameters: match 10, mismatch −3, gap opening penalty −50, gap extension penalty −5, and longest penalized gap 10; the terminal gaps are not penalized at all. A window size of 20 bp was used with a step of 10 bp. The analysis window was moved upstream and downstream of start and stop codons, respectively. The normalized score for a given window is calculated as a fraction of an average score for all UTRs in a given window over the maximum score observed in all 5′ or 3′ UTRs, respectively. Untranslated region replacement A replacement of the entire UTR may lead to drastic changes in gene expression, especially if a UTR having a posttranscriptional signal is replaced by another. We compared the evolutionary distances of UTRs between primate and rodent orthologous sequences. We based our analysis on the UTR sequence distances that contradicted the expected phylogenetic tree of relatedness. We could detect 149 UTR replacements distributed among different species. Some of the observed replacements may result from selection of different AS isoforms of a single locus in different species. This is particularly likely if an AS event involves an alternative first or last exon. It seems that UTR replacements are more frequent in rodents than in primates, but the difference is not statistically significant at the 5% significance level ( Table S9 ). We detected a UTR replacement in less than 2% of the analyzed sequences. The evolutionary consequences could be significant because the UTR replacement might result in changes in expression level or the loss of an mRNA localization signal. The H-Invitational Database All the results of the mapping of the FLcDNA sequences onto the human genome, the clustering of FLcDNA sequences, sequence alignments, detection of AS transcripts, sequence similarity searches, functional annotation, protein structure prediction, subcellular localization prediction, SNP mapping, and evolutionary analysis, as well as the basic features of FLcDNA sequences, are stored in the H-InvDB ( Figure S8 ). The H-InvDB is a unique database that integrates annotation of sequences, structure, function, expression, and diversity of human genes into a single entity. It is useful as a platform for conducting in silico data mining. The database has functions such as a keyword search, a sequence similarity search, a cDNA search, and a searchable genome browser. It is hoped that the H-InvDB will become a vital resource in the support of both basic and applied studies in the fields of biology and medicine. We constructed two kinds of specialized subdatabases within the H-InvDB. The first is the Human Anatomic Gene Expression Library (H-Angel), a database of expression patterns that we constructed to obtain a broad outline of the expression patterns of human genes. We collected gene expression data from normal and diseased adult human tissues. The results were generated using three methods on seven different platforms. These included iAFLP ( Kawamoto et al. 1999 ; Sese et al. 2001 ), DNA arrays (long oligomers, short oligomers [ Haverty et al. 2002 ], cDNA nylon microarrays [ Pietu et al. 1999 ], and cDNA glass slide microarrays [Arrays/IMAGE-Genexpress]), and cDNA sequence tags (SAGE [ Velculescu et al. 1995 ; Boon et al. 2002 ], EST data [ Boguski et al. 1993 ; Kawamoto et al. 2000 ], and MPSS [ Brenner et al. 2000 ]). By normalizing levels of gene expression in experiments conducted with different methods, we determined the gene expression patterns of 19,276 H-Inv loci in ten major categories of tissues. This analysis allowed us to clearly distinguish broadly and evenly expressed housekeeping genes from those expressed in a more restricted set of tissues (details will be published elsewhere). The H-Angel database comprises the largest and most comprehensive collection of gene expression patterns currently available. Also provided is a classification of human genes by expression pattern. The second subdatabase of the H-InvDB is DiseaseInfo Viewer. This is a database of known and orphan genetic diseases. We tried to relate H-Inv loci with disease information in two ways. Firstly, 613 H-Inv loci that correspond with known, characterized disease-related genes were identified by creating links to entries in both LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) and OMIM ( Hamosh et al. 2002 ). To explore the possibility that cDNAs encoding unknown proteins may be related to “orphan pathologies” (diseases that have been mapped to chromosomal regions, but for which associated genes have not yet been described), we generated a list of H-Inv loci that co-localized with these cytogenetic regions. The nonredundant orphan disease dataset we created consists of 586 diseases identified through OMIM ( http://www.ncbi.nlm.nih.gov/Omim/ , ver. Jan. 2003), with an additional 108 identified from GenAtlas ( http://www.dsi.univ-paris5.fr/genatlas/ , ver. Jan. 2003). Using the OMIM and GenAtlas databases in conjunction with the annotation results from the H-InvDB may accelerate the process of identifying candidate genes for human genetic diseases. Concluding Remarks There are a number of established collections of nonhuman cDNAs, such as those of Drosophila melanogaster ( Stapleton et al. 2002 ), Danio rerio ( Clark et al. 2001 ), Arabidopsis thaliana ( Seki et al. 2002 ), Plasmodium falciparum ( Watanabe et al. 2002 ), and Trypansoma cruzi ( Urmenyi et al. 1999 ). The most extensive collection of mammalian cDNAs so far has been that of the RIKEN/FANTOM mouse cDNA project ( Kawai et al. 2001 ; Okazaki et al. 2002 ). This wealth of information has spurred a wide variety of research in the areas of both gene expression profiling ( Miki et al. 2001 ) and protein–protein interactions ( Suzuki et al. 2001 ). The H-InvDB provides an integrative means of performing many more such analyses based on human cDNAs. The most important findings that have resulted from the cDNA annotation are summarized here. (1) The 41,118 H-Inv cDNAs were found to cluster into 21,037 human gene candidates. Comparison with known and previously predicted human gene sets revealed that these 21,037 hypothesized gene clusters contain 5,155 new gene candidates. (2) The primary structure of 21,037 human gene candidates was precisely described. For the majority of them we observed that both first introns and last exons tended to be longer than the other introns and exons, respectively, implying the possible existence of intriguing mechanisms of transcriptional control in first introns. (3) We discovered the existence of 847 human gene candidates that could not be convincingly mapped to the human genome. This result suggested that up to 3.7%–4.0% of the human genome sequences (NCBI build 34 assembly) may be incomplete, containing either unsequenced regions or regions where sequence assembly has been performed in error. (4) Based on H-Inv cDNAs, we were able to define an experimentally validated AS dataset. The dataset was composed of 3,181 loci that encoded a total of 8,553 AS isoforms. In the 55% of ORFs containing AS isoforms, the pattern of alternative exon usage was found to encode different functional domains at the same loci. (5) A standardized method of human curation for the H-Inv cDNAs was created under the tacit consensus of international collaborations. Using this method, we classified 19,574 H-Inv proteins into five categories based on sequence similarity and structural information. We were able to assign functional definitions to 9,139 proteins, to locate function- or family-defining InterPro domains in 2,503 further proteins, and to identify 7,800 transcripts as good candidates for hypothetical proteins. (6) A total of 1,892 H-Inv proteins were assigned identities as one of 656 different EC-numbered enzymes. This enzyme library includes 32 newly identified human enzymes on known metabolic pathway maps and comprises the largest collection of computationally validated human enzymes. (7) Based on a variety of supporting evidence, 6.5% of H-Inv loci (1,377 loci) do not have a good protein-coding ORF, of which 296 loci are strong candidates for ncRNA genes. (8) We identified and mapped 72,027 SNPs and indels to unique positions on 16,861 loci. Of these, 13,215 nonsynonymous SNPs, 358 nonsense SNPs, and 452 indels were found in coding regions and may alter protein sequences, cause phenotypic effects, or be associated with disease. In addition, we identified 216 polymorphic microsatellite repeats on 213 loci, 25 of which were located in coding regions. (9) During human proteome analysis, it was suggested that the basic gene set of humans might have been established in the early stage of animal evolution. Our analysis of UTRs revealed that insertions or deletions near coding regions were rare when compared with substitutions, though in some cases drastic changes such as UTR replacements occurred. (10) A consequence of the annotation process and our related research was the development of the H-InvDB to contain our annotation work. H-InvDB is a comprehensive database of human FLcDNA annotations that stores all information produced in this project. As a subdivision of H-InvDB, we developed two other specialized subdatabases: H-Angel and DiseaseInfo Viewer. H-Angel is a database of gene expression patterns for 19,276 loci. DiseaseInfo Viewer is a database of known disease-related genes and loci co-localized with 694 orphan pathologies. These pathologies were mapped onto the genome but were not identified experimentally. In the H-Inv project, we collected as many FLcDNAs as possible and conducted extensive analyses concerning the quality of cDNAs, such as detection of frameshift errors, retained introns, and internal poly-A priming, under a unified criterion. Although these analyses are still in an elementary state, we store these results in H-InvDB to share this information with the biological community. We believe that this is an important contribution of our project, because it will provide a reliable way to control the quality of the cDNA clones. In the future, this information will be useful for improving the methods of clone library construction. It has been suggested that the human genome encodes 30,000 to 40,000 genes. In this study we comprehensively evaluated more than 21,000 human gene candidates (up to 70% of the total). Thus, efforts should be continued by the H-Inv consortium and others to “fully” characterize the human transcriptome. For this purpose new technologies should be implemented that are more sensitive in detecting rarely expressed genes and AS transcripts. Nevertheless, there are unavoidable limitations for human cDNA collections, such the identification of embryo-specific genes, for which other approaches should be employed. One alternative is the use of ab initio predictions from genomic sequences, in conjunction with expression profiling studies, to identify rarely expressed genes that share structural similarity to known genes. Additionally, a better characterization of cis -regulatory element units may help to define the boundary of other genes that are undetected by current gene prediction programs. Another area that remains to be explored is the identification of potential hidden RNA gene families that may play vital roles, such as the recently uncovered family of microRNA genes, which is involved in the regulation of expression of other genes (for review see Ambros 2001 ; Moss 2002 ). The proteome determination aspects of this project, including the identification of new enzymes and hypothetical proteins, should stimulate more focused biochemical studies. The functional classifications may allow definition of subproteomes that are related to different physiological processes. The H-Inv transcriptome based on the definition of a consensus proteome (the H-Inv proteins) links both the analysis of genomic DNA and direct proteome analysis with the study of expressed mRNA analysis from different tissues, cells, and disease states. It creates a standard for the comparison of disease-related alterations of the human proteome. Moreover, comparison with pathogen proteomes may yield many possible drug target proteins. Also, the annotation of ncRNAs raises the possibility of novel “smart” therapeutics that could either inhibit or mimic the mechanisms of these RNAs. The H-Inv project is the first ever comprehensive compilation of curated and annotated human FLcDNAs. The project may lead to a more complete understanding of the human transcriptome and, as a result, of the human proteome. The preceding examples of the importance of the H-Inv data in understanding human physiology and evolution represent just a small fraction of the research potential of the H-InvDB. In conclusion, the H-InvDB platform constructed to hold the results of the comprehensive annotations performed by our international team of collaborators represents a substantial contribution to resources that are needed for further exploration of both human biology and pathology. Materials and Methods cDNA resources 41,118 H-Inv cDNAs were sequenced by the Human Full-Length cDNA Sequencing Project ( Ota et al. 1997 ; Yudate et al. 2001 ; Ota et al. 2004 ) at the Helix Research Institute, the Institute of Medical Science at the University of Tokyo, and the Kazusa DNA Research Institute (20,999 sequences in total); the Kazusa cDNA Sequencing Project ( Kikuno et al. 2002 ) at the Kazusa DNA Research Institute (2,000 sequences); the Mammalian Gene Collection ( Strausberg et al. 1999 ) at the National Institutes of Health in the United States (11,806 sequences); the German Human cDNA Project ( Wiemann et al. 2001 ) coordinated by the Deutsches Krebsforschungszentrum in Heidelberg (5,555 sequences); and the Chinese National Human Genome Center at Shanghai (Hu et al. 2000) (758 sequences). Mapping human cDNAs to the human genome and the comparison of the mapped H-Inv cDNAs with other annotated datasets We have mapped human cDNA sequences to the human genome sequence corresponding to the NCBI build 34 assembly. The datasets we used were a set of 41,118 H-Inv cDNAs and a set of 37,488 human RefSeq sequences available on 15 July 2002 and on the 1 September 2003, respectively. All the revisions for H-Inv cDNA sequences until August 2003 were applied in the datasets. Before performing the mapping procedure, all the repetitive and low-complexity sequences in all the cDNA sequences were masked using RepeatMasker ( http://ftp.genome.washington.edu/RM/RepeatMasker.html ) and Repbase 7.5. Then we used the cross_match program to mask the remaining vector sequences in each cDNA sequence. Any poly-A tails were also masked by using a custom-made Perl script. In the first step of the mapping procedure, we conducted BLASTN (ver.2.2.6) searches of all the sequences against the human genome sequence and extracted the corresponding genomic regions for each query sequence. Then we used est2genome (EMBOSS package ver.2.7.1) to align each sequence to the genomic region with a threshold of 95% identity and 90% coverage. Coverage of each cDNA sequence was calculated excluding those from the vector and poly-A tails that were masked in the previous step. If the sequences were mapped to multiple positions on the human genome, then we selected their best locus based on the identity, length coverage, and number of exons of those sequences. As a result, 77,315 sequences (including 40,140 cDNAs from the H-Inv project) were successfully mapped onto the human genome and were clustered into 38,587 clusters based on sharing at least 1 bp of an exon on the same chromosome strand. We used all the mapped sequences, including human RefSeq sequences, to compare the clusters that included H-Inv cDNAs with those that consisted of only human RefSeq sequences. 20,190 clusters out of 38,587 consisted of only H-Inv cDNAs or both H-Inv cDNAs and human RefSeq sequences. The rest of the clusters consisted of RefSeq sequences only. All of the mapped cDNAs and the overlap with the RefSeq sequences can be viewed using G-integra in the H-InvDB ( http://www.jbirc.aist.go.jp/hinv/g-integra/html/ ). The mapping procedure for all the unmapped cDNAs against the mouse genome was also performed, using a threshold of 60% identity and 90% coverage. Clustering of unmapped sequences The sequences that were not mapped onto the human genome were clustered by a single linkage clustering method. The similarity search was performed among all the unmapped sequences. The program used was MegaBLAST version 2.2.6 ( Zhang et al. 2000 ). As with to the mapping strategy, some distinctive sequences (repetitive regions, contaminations from cloning vectors and poly-A tails) were excluded from the queries of the similarity search. The similarity was evaluated using the expected value ( E- value) between two sequences. Only when the E- value of the two sequences was calculated to be 0, did we assume that a significant level of similarity was detected between the two sequences. Identification of gene structure In order to identify gene structure, we used only the representative H-Inv cDNAs. When detecting repetitive elements in cDNAs, RepeatMasker was conducted in a similar manner to the previous phase. We used curated cDNAs in which frameshift errors and remaining introns were removed. Prediction of ORFs We predicted ORFs in all 41,118 H-Inv cDNAs, as illustrated in Figure S1 , based on the alignment of similarity searches by FASTY ( Pearson 2000 ; Mackey et al. 2002 ) (ver. 3.4t11) and BLASTX ( Altschul et al. 1990 ) (ver. 2.0.11), and gene prediction by GeneMark ( McIninch et al. 1996 ) ( http://opal.biology.gatech.edu/GeneMark/ ) ( Table S10 ). Prior to the prediction of ORFs, we judged if the sequence had any frameshift errors or remaining introns (see Figure S1 ). During ORF prediction, we corrected the aforementioned sequence irregularities computationally. Procedure of computational and human annotation Prior to the human curation, we performed two computational automated annotation processes to select the representative clone for each locus and to predict function of H-Inv proteins (see Figure S2 ). We then assigned the most suitable data source ID to each H-Inv protein following a scheme illustrated in Figure S2 and referring to the information using newly developed annotation viewers, named SOUP location viewer, SOUP annotation viewer, and Similarity Motif ORF (SMO) Viewer ( Figure S9 ). Questionable transcripts were determined by human curation based upon evidence such as the following: sequences with no similarity to a known protein or domain, sequences with a very short ORF, cDNAs with only a single exon, and sequences with no EST support. Only 959 (4.9%) of the computationally selected 19,574 representative H-Inv proteins had to be manually corrected. Another 3,142 (16.1%) of the H-Inv proteins had their functional assignment altered by manual curation. Assignment of functional motifs Nonredundant proteome datasets were obtained for fly ( http://flybase.bio.indiana.edu/ ), worm ( http://www.wormbase.org/ ), budding yeast ( http://www.pasteur.fr/externe ), fission yeast ( http://www.sanger.ac.uk/ ), plant ( http://mips.gsf.de/proj/thal/index.html ), and a bacteria ( ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Escherichia_coli_K12/ ). The H-Inv proteins and other nonredundant proteome datasets were assigned InterPro codes by InterProScan ver. 3.1 ( Mulder et al. 2003 ). The codes corresponded to families, domains, and repeats. GO terms were also assigned (see Table S5 ). Evolutionary relationship of proteomes The top 40 InterPro entries for the human proteome were compared with their equivalents from the fly, worm, yeasts, plant, and bacteria proteomes (see Table S4 ). Protein domains and low-complexity inserted sequences Folds were assigned by reverse PSI-BLAST ( Altschul et al. 1997 ) searches of the amino acid sequences derived from the H-Inv cDNA against the SCOP database ( Lo Conte et al. 2000 ). Information on protein and gene structures, with the exception of mouse and puffer fish, was obtained from the individual genome projects ( Blattner et al. 1997 ; Kunst et al. 1997 ; CESC 1998 ; Adams et al. 2000 ; AGI 2000 ; Wood et al. 2002 ). The data for mouse and puffer fish were obtained from the Ensembl database ( Hubbard et al. 2002 ). Subcellular localization Subcellular localization targeting signals and transmembrane helices of 40,352 H-Inv proteins were predicted using the PSORT II ( Nakai and Horton 1999 ), TargetP ( Emanuelsson et al. 2000 ), TMHMM, and SOSUI ( Hirokawa et al. 1998 ) computer programs. UTR sequences We obtained the UTR sequences from three primates ( Pan troglodytes, chimpanzee; Macaca fascicularis, crab-eating macaque; and Macaca mulatta, rhesus monkey) and two rodents ( Mus musculus, house mouse; and Rattus norvegicus, Norwegian rat) that corresponded to UTRs from Homo sapiens . In order to do this, we mapped the cDNAs to the human or mouse genome. The corresponding rodent cDNAs were determined by using a human–mouse genome alignment provided by Ensembl. cDNAs of the primates and rodents were retrieved from the DDBJ/EMBL/GenBank databases using the cut off date of 15 July 2002. Additionally, we used the FANTOM2 mouse sequences released on 5 December 2002, and 4,063 5′ ESTs of chimpanzees ( Sakate et al. 2003 ). Corresponding UTRs between human and other species were identified by aligning 5′ and 3′ ends of the human ORFs. To compare evolutionary distances, we analyzed 3,061 and 5,277 orthologous groups that consisted of at least three species' information for the 5′ and 3′ UTR sequences, respectively. Supporting Information Dataset S1 List of Library Origins of H-Inv cDNAs (182 Libraries) The dataset consists of 41,118 H-Inv cDNAs that were cloned from cDNA libraries derived from 182 varieties of cell and tissue. (33 KB XLS). Click here for additional data file. Dataset S2 List of H-Inv Proteins with Potential EC Numbers (1,892 H-Inv Proteins) The allotted EC numbers are based on the corresponding DNA databank records, UniProt/Swiss-Prot and TrEMBL records that show sequence similarity to the proteins, and InterPro records that the proteins hit. (247 KB XLS). Click here for additional data file. Dataset S3 List of Polymorphic Microsatellites Inferred by Comparisons between the H-Inv cDNAs and Genomic Sequences (56 KB XLS). Click here for additional data file. Figure S1 Prediction of ORFs (A) Schematic diagram for the prediction of ORFs. This diagram illustrates the ORF prediction method used on all H-Inv cDNAs. The method was based upon the alignment of similarity searches using FASTY and BLASTX. Gene prediction was carried out using GeneMark. Prior to the prediction of ORFs, we judged if a sequence had any frameshift errors or remaining introns. During ORF prediction, we corrected those sequence irregularities computationally. Details of how sequence irregularities were predicted are described in (B) and (C). (B) Schematic diagram for prediction of unspliced introns. This schematic diagram illustrates the prediction method used for unspliced introns. (C) Schematic diagram for prediction of frameshift errors. Frameshift errors were inferred from cDNA–genome pairwise alignment gaps due to insertion or deletion, exception of multiple of 3 bp, or over 10 bp in either the query cDNA or genome. (D) The statistics for the predicted frameshifts and unspliced introns. (49 KB PDF). Click here for additional data file. Figure S2 Scheme of Prediction for Functional Annotation (A) Schematic diagram for determining a representative transcript for each locus. The procedure of computational autoannotation is illustrated. Prior to the human curation of the representative transcript of each H-Inv cluster, we performed computational autoannotation. (B) Schematic diagram for functional prediction of H-Inv proteins. This schematic diagram illustrates the H-Inv autofunctional annotation pipeline that can determine the most appropriate data source ID, avoiding the following keywords that suggest proteins without experimental verification in the description; (1) hypothetical, (2) similar to, (3) names of cDNA clones (Rik, KIAA, FLJ, DKFZ, HSPC, MGC, CHGC, and IMAGE) and (4) names of InterPro domain frequent hitters. (34 KB PDF). Click here for additional data file. Figure S3 Size Distribution of Predicted ORFs The size distribution of all H-Inv proteins among the five similarity categories. (24 KB PDF). Click here for additional data file. Figure S4 Features of Category II Proteins A total of 4,104 H-Inv proteins were classified as Category II based on sequence similarity to functionally validated proteins. The table and figure show source species of proteins in public databases to which the Category II proteins were similar. (9 KB PDF). Click here for additional data file. Figure S5 H-Inv KEGG Analysis Results (Images of KEGG Pathways) The images illustrate the metabolic pathways of KEGG database based on the EC number assignments to H-Inv proteins. (47 KB PDF). Click here for additional data file. Figure S6 Numbers of Representative H-Inv cDNAs That Are Homologous to Proteins in Each Taxonomic Group Two thresholds (E < 10 −5 , white bars, and E < 10 −10 , black bars) were employed. The “animal” group does not include mammalian species. The “eukaryote” group represents eukaryotic species other than animals, fungi, and plants. (9 KB PDF). Click here for additional data file. Figure S7 A Functional Classification of H-Inv Protein Families That Have Homologs in Each Taxonomic Group H-Inv protein families were identified by clustering H-Inv proteins using the single-linkage clustering method. Then, the number of homologs for each H-Inv protein family was calculated. Mammalian species are excluded from the “animal” group. “eukaryote” represents eukaryotic species other than animals, fungi, and plants. Single-linkage clustering . All of the H-Inv proteins were compared with themselves by BLASTP and clustered with the thresholds of E-values of 10 −30 and 10 −50 . The numbers of singleton families detected were 11,890 and 13,938 at the E-value of 10 −30 and 10 −50 , respectively. (49 KB PDF). Click here for additional data file. Figure S8 A Sample View of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/ ) A FLcDNA (BC003551) is shown with its detailed annotations, e.g., gene structure, functional annotation, ORF predictions, protein structure prediction by GTOP, etc. The H-InvDB has links to other internal databases (red boxes) such as a genome map viewer (G-integra) and gene expression library (H-Angel). Green boxes show internal viewers for the results of clustering (Clustering Viewer showing results by H-Inv, STACK, TIGR, UniGene, etc.), the prediction of subcellular localization (TOPOViewer showing results of TMHMM, SOSUI, TargetP, and PsortII), and the disease-related information (DiseaseInfo Viewer linking to OMIM and GenAtlas). The H-InvDB also has links to many external public databases (black boxes), including DDBJ/EMBL/GenBank, RefSeq, UniProt/Swiss-Prot and TrEMBL, Genew, InterPro, 3D Keynote, Ensembl, GeneLynx, LocusLink, PubMed, LIFEdb, dbSNP, GO, and GTOP, and to homepages by original data producers of FLcDNA clones and sequences (blue boxes), including the Chinese National Human Genome Center (CHGC), the Deutsches Krebsforschungszentrum (DKFZ/MIPS), Helix Research Institute (HRI), the Institute of Medical Science at the University of Tokyo (IMSUT), the Kazusa DNA Research Institute (KDRI), the Mammalian Gene Collection (MGC/NIH), and the FLJ project. (2,650 KB PDF). Click here for additional data file. Figure S9 H-Inv Annotation Viewers (A) G-integra: A genome mapping viewer. (B) SOUP Locus annotation viewer. (C) SOUP cDNA annotation viewer. (D) SMO Viewer: The similarity, motif, and ORF information viewer. (2,022 KB PDF). Click here for additional data file. Table S1 Gene Structure (A) Gene structure of the cDNAs. (B) The frequencies and varieties of repetitive sequences found in the cDNAs. A list of the 20,899 loci representing cDNAs that RepeatMasker showed contained repetitive elements. (C) The positions (5′ UTR, ORF, and 3′ UTR) of repetitive sequences in the protein-coding cDNAs. A total of 1,863 cDNAs contained repetitive sequences in their ORF, of which 549 had repetitive sequences within their most probable ORF. Repetitive sequences appeared in 2,240 and 5,401 cDNAs in their 5′ UTRs and 3′ UTRs, respectively. (20 KB PDF). Click here for additional data file. Table S2 CAI and Codon Usage (A) CAI was measured for all H-Inv proteins. CAI is a measure of biased patterns for synonymous codon usage ( http://biobase.dk/embossdocs/cai.html ). (B) Codon usage in predicted ORFs of H-Inv proteins. Total tri-nucleotide frequencies (forward strand) for the sequences of each species are shown. Nonredundant proteome datasets for nonhuman species were obtained from the following sites: fly ( Drosophila melanogaster ; http://flybase.bio.indiana.edu/ ), worm ( Caenorhabditis elegans ; http://www.wormbase.org/ ), budding yeast ( Saccharomyces cerevisiae ; http://www.pasteur.fr/externe ), fission yeast ( Schizosaccharomyces pombe ; http://www.sanger.ac.uk/ ), plant ( Arabidopsis thaliana ; http://mips.gsf.de/proj/thal/index.html ), and bacteria ( Escherichia coli K12; ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Escherichia_coli_K12/ ). (20 KB PDF). Click here for additional data file. Table S3 Tissue Library Origins of H-Inv Proteins The results of classification into five similarity categories for each of ten tissue classes. (A) Numbers of H-Inv proteins. (B) Histogram. (10 KB PDF). Click here for additional data file. Table S4 The InterPro IDs Identified in H-Inv Proteins The top 40 InterPro IDs identified in H-Inv proteins and proteins from other species are listed for all types (A) and for each type of family, domain, and repeat (B–D). Analyses were conducted by InterPro ver. 3.1. Nonredundant proteome datasets of other species were obtained from the following sites: fly ( Drosophila melanogaster ; http://flybase.bio.indiana.edu/ ), worm ( Caenorhabditis elegans ; http://www.wormbase.org/ ), budding yeast ( Saccharomyces cerevisiae ; http://www.pasteur.fr/externe ), fission yeast ( Schizosaccharomyces pombe ; http://www.sanger.ac.uk/ ), plant ( Arabidopsis thaliana ; http://mips.gsf.de/proj/thal/index.html ), and bacteria ( Escherichia coli K12; ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Escherichia_coli_K12/ ). (36 KB PDF). Click here for additional data file. Table S5 GO Term Assignment to H-Inv Proteins (A) Molecular function. (B) Cellular component. (C) Biological process. (74 KB PDF). Click here for additional data file. Table S6 List of Newly Assigned Human Enzymes (32 H-Inv Proteins) All these 32 H-Inv proteins were newly assigned enzyme numbers with the support of the KEGG pathway. These enzyme assignments were previously unrepresented in Homo sapiens. (33 KB PDF). Click here for additional data file. Table S7 A Functional Classification of Representative H-Inv cDNAs That Have Homologs in Other Species (See also Figure 6 .) (9 KB PDF). Click here for additional data file. Table S8 Basic Statistics for UTR Sequences Analyzed (8 KB PDF). Click here for additional data file. Table S9 UTR Replacements in Primates and Rodents One hundred and forty-seven UTR replacements distributed among different species were detected. (9 KB PDF). Click here for additional data file. Table S10 List of the Databases and Software Used in the H-Inv Project (31 KB PDF). Click here for additional data file. Protocol S1 A Detailed Functional Annotation Based on Protein Modules (25 KB PDF). Click here for additional data file. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC393292.xml |
544593 | Echocardiographic AV-interval optimization in patients with reduced left ventricular function | Background Ritter's method is a tool used to optimize AV delay in DDD pacemaker patients with normal left ventricular function only. The goal of our study was to evaluate Ritter's method in AV delay-interval optimization in patients with reduced left ventricular function. Methods Patients with implanted DDD pacemakers and AVB III° were assigned to one of two groups according to ejection fraction (EF): Group 1 (EF > 35%) and Group 2 (EF < 35%). AV delay optimization was performed by means of radionuclide ventriculography (RNV) and application of Ritter's method. Results For each of the patients examined, we succeeded in defining an optimal AV interval by means of both RNV and Ritter's method. The optimal AV delay determined by RNV correlated well with the delay found by Ritter's method, especially among those patients with reduced EF. The intra-class correlation coefficient was 0.8965 in Group 1 and 0.9228 in Group 2. The optimal AV interval in Group 1 was 190 ± 28.5 ms, and 180 ± 35 ms in Group 2. Conclusion Ritter's method is also effective for optimization of AV intervals among patients with reduced left ventricular function (EF < 35%). The results obtained by RNV correlate well with those from Ritter's method. Individual programming of the AV interval is fundamentally essential in all cases. | Background Since introduction of the DDD pacemaker in the early 1980s, researchers have repeatedly attempted to optimize the atrioventricular (AV) interval, for the purpose of maximizing patient hemodynamic performance. Cannon waves may be induced by programming excessively short AV intervals, and diastolic mitral regurgitation may occur with excessively long programmed AV intervals. The AV interval is considered optimal (AV opt ) if it allows maximum cardiac output. The duration of the optimal AV interval varies throughout a wide range among individuals, primarily the result of appreciable differences in interatrial conduction [ 1 - 4 ]. An extensive variety of techniques has been employed to optimize AV delay, including acquisition and analysis of essential hemodynamic parameters by means of aortic-valve Doppler signals, impedance cardiography [ 9 - 11 ], Swan-Ganz catheterization [ 12 - 15 ], and especially the stroke volume [ 5 - 8 ]. Leman et al. [ 16 ] have demonstrated that it is also possible to utilize measurement of left ventricular ejection fraction and stroke volume by myocardial thallium scintigraphy as a means of AV interval optimization. A further possibility involves detection of left atrial depolarization by an esophageal electrode recording [ 17 , 18 ]. During recent years, the use of Doppler echocardiography in conjunction with the mitral valve inflow profile has been investigated as means of AV interval optimization: i.e., Ritter's method [ 19 ]. Previous investigations have evaluated Ritter's method in patients with normal left ventricular ejection fractions. During recent years, cardiac resynchronization therapy (CRT) has increasingly gained in significance for patients with chronic heart failure (CHF) [ 20 ]. In cases without ventricular desynchronicity, normal DDD pacemakers (or ICDs with DDD pacemaker function) will in future continue to be implanted in patients with reduced left ventricular ejection fraction. The goal of our study was accordingly to apply Ritter's method – until now validated only for patients with normal EF – for patients with reduced left ventricular ejection fraction (EF < 35%). Methods We studied 20 DDD pacemaker patients within the context of in-office follow-up. Table 1 shows the baseline characteristics and Table 2 , the inclusion criteria. We classified patients into two groups, according to left ventricular ejection fraction results obtained by echocardiography. Group 1 consisted of 10 patients with normal left ventricular ejection fraction, or with moderately reduced EF (EF > 35%). Group 2 comprised 10 patients with appreciably reduced left ventricular ejection fraction (EF < 35%). Table 1 Clinical characteristics of the patients Characteristics Group 1 (EF > 35%) ( n = 10) Group 2 (EF < 35%) ( n = 10) Age 68.5 ± 4.5 65.7 ± 6.3 Male sex (%) 60 100 Left ventricular ejection fraction (%) 58 ± 9.7 22 ± 7.4 Left ventricular end-diastolic dimension (mm) 47 61 Coronary artery disease (%) 30 50 Dilated cardiomyopathy (%) 0 50 Hypertension (%) 30 0 Pharmacologic therapy (%) ACE inhibitor 40 100 Beta-blocker 50 90 Loop diuretic 0 100 Spironolactone 0 70 Table 2 Inclusion criteria DDD pacemaker by AVB III° with permanent atrial and ventricular pacing No left bundle-branch block or possible indication for CRT Pacemaker implantation at least 4 months beforehand NYHA Class I or II We performed ejection fraction analysis by RNV and Ritter's method to achieve AV optimization, for 5 AV intervals in the range of 100 to 250 ms. We performed all measurements within 15 minutes of AV interval programming for every patient. All patients were permanently stimulated in the right atrium and right ventricle (binodal disease). Patient heart rate remained constant during the measurement period at programmed pacemaker lower rate (60 – 70 beats/min). Analysis of left ventricular ejection fraction by RNV We performed radionuclide ventriculography (RNV) after in vivo marking of erythrocytes with tin DTPA and 10 MBq/kg KG Tc-99m, using a single-head Gamma camera (CGR gammatome 2, General Electrics, Paris, France) with a high-resolution, medium-energy collimator. For RNV we applied the equilibrium technique at 16 frames per cycle with patients at rest, and ventricular pacing at programmed AV intervals. We acquired a minimum of one million counts per image, and stored the data in a 64 × 64 matrix. We calculated left ventricular ejection fraction (LVEF) semi-automatically after spatial and temporal smoothing and background subtraction. After Fourier analysis of ventricular stimulation progression, we recorded (with examiner definition) a region of interest (ROI) around the end-diastolic contour of the left ventricle and calculated the LVEF as follows: Ritter's method By 1994 a method developed by Ritter et al. had become established for optimizing the AV interval [ 19 ]. A prerequisite for application of Ritter's method is Doppler-echocardiographic measurement of the mitral inflow profile. Ritter's method employs the following formula for calculation of the optimal AV interval: AV opt = AV long - ( a - b ) We applied the following procedure in application of this formula (see Fig. 1 ): Figure 1 Ritter's method: The first step is determination of "a" for a nonphysiologically short AV interval (e.g. 125 ms), followed be determination of "b" for a nonphysiologically long AV interval (e.g. 250 ms). Step 1 The first step involves programming for the pacemaker a nonphysiologically short AV interval, followed by determination of "a". This value "a" is the temporal interval between the ventricular contraction spike and the end of the A wave. "a" designates the electromechanical delay between right ventricular stimulation and the beginning of the left ventricular systole (i.e., closure of the mitral valve). Step 2 The next step is programming for the pacemaker a long AV interval ( AV long ), followed by determination of "b". This value "b" is the temporal interval between the ventricular contraction spike and the end of the A wave. AV long - b defines the duration of the undisturbed maximal diastolic left ventricular filling. The purpose of AV interval optimization in accordance with Ritter is to allow the ventricular systole to begin immediately subsequent to maximum, undisturbed diastolic ventricular filling and, in turn to prevent the occurrence of Cannon waves as well as diastolic mitral regurgitation. Statistics We applied intra-class correlation in performing statistical evaluation. Results Group 1 In a given patient, our results indicated that it was possible to define an optimal AV interval for every patient: both by RNV as well as by Ritter's method. The mean optimal AV interval was 190 ± 28.5 ms. The correlation between RNV and Ritter's method is good: the intra-class quotient is 0.8965 (see Fig. 2 ). In results calculated by RNV, the mean percent difference in left ventricular ejection fraction between the hemodynamically best and worst AV intervals was 11 ± 4% (see Fig. 4 ). Figure 2 The correlation of the results of the RNV and Ritter methods, with respect to the optimal AV interval for Group 1. Figure 4 The maximum difference in left ventricular EF, determined by RNV and as a function of the programmed AV interval, for each of the patients examined. Group 2 In a given patient, we likewise succeeded in defining the optimal AV interval for every patient: both by RNV as well as by Ritter's method. The mean optimal AV interval was 180 ± 35 ms. In Group 2 as well, there was good correlation between RNV and Ritter's method: the intra-class quotient was 0.9228 (see Fig. 3 ). In results calculated by RNV, the mean percent difference in left ventricular ejection fraction between the hemodynamically best and worst AV intervals was 28 ± 11% (see Fig. 4 ). Figure 3 The correlation of the results of the RNV and Ritter methods, with respect to the optimal AV interval for Group 2. Discussion A number of studies have documented the importance of AV synchronization for maximizing the left ventricular ejection fraction in pacemaker patients [ 21 - 24 ]. Despite CRT, the implantation of a DDD pacemaker (or ICD with DDD-pacemaker function) is still justified for patients with reduced left ventricular function and a lack of ventricular asynchrony. The goal of our study was accordingly to apply Ritter's method for patients with reduced left ventricular ejection fraction. In every subject of Groups 1 and 2, it was possible on the basis of the left ventricular ejection fraction to define the optimal AV delay by means of RNV. The method of AV delay optimization by RNV has been previously verified [ 16 ]. The cost and complexity of this method, however, have hindered its extensive clinical application. On the basis of minimal inter- and intraobserver variability this method is nevertheless very well suited as a reference method. Our application of Ritter's method enabled definition of the optimal AV interval for all patients. We further determined that Ritter's method can be reliably employed even in cases of reduced left ventricular systolic function. The AV interval calculated by Ritter's method correlated well with data obtained by RNV: both for normal (with intra-class coefficient of 0.8965) as well as for reduced left ventricular EF (intra-class coefficient of 0.9228). Since Ritter's initial publication in 1994, AV interval optimization on the basis of the mitral valve inflow profile has been reported in one additional study [ 19 ]. In 1997 Kindermann et al. compared results calculated from Ritter's formula with those obtained from impedance cardiography [ 10 ]. This study established a high degree of correlation between the results for the optimal AV interval determined by the two different methods. The mean deviation in optimal AV interval between the results from Ritter's formula and determination of stroke volume by impedance cardiography was ± 26 ms for the atrial-triggering mode, and ± 30 ms for the AV sequential mode. Kindermann et al. criticized the fact that it is possible to apply Ritter's method only for patients with ventricular stimulation. In comparison to time-consuming and expensive RNV, and AV-interval optimization by Swan-Ganz catheterization (with the associated risks of an invasive procedure), Ritter's method offers the following advantages: it is non-invasive and can be quickly performed (approx. 5 min.). It does not require long years of echo experience, and it is cost-effective. Even with patients not readily amenable to sonographic detection, the mitral valve inflow profile is almost always qualitatively satisfactory enough to allow application of Ritter's method. The only noteworthy disadvantage of this method is the necessity for continuous ventricular stimulation: which means that it can be used only for patients with a complete AV block. Patients with only intermittent high-grade AV blocks are accordingly not suited for Ritter's method. In our patients, the mean optimal AV interval in Group 1 (EF > 35%) was 190 ± 28.5 ms. In comparison, the optimal AV interval among the patients with chronic heart failure in Group 2 was 180 ± 35 ms. Data in the literature are not consistent on the duration of the optimal AV delay. Kindermann [ 10 ] considers AV opt = 88 ms ± 35 ms with atrial triggering, and AV opt = 143 ms ± 41 ms for the AV sequential mode. Knorre [ 18 ] has determined AV opt = 100.5 ± 27.8 ms for atrial triggering, and AV opt = 169 ± 24.5 ms for the AV sequential mode. Haskel [ 5 ] has established the best AV interval to be 150 ms. Janosik [ 6 ] considers AV opt = 144 ± 48 ms with atrial triggering, and AV opt = 176 ± 44 ms for the AV sequential mode. Ishikawa [ 15 ] has determined AV opt = 161 ± 26 ms. Our results on the length of the optimal AV delay lie within the range found in the literature. The variance in data observed in some cases emphasizes the highly individual nature of the optimal AV delay: indeed, it results from the interatrial conduction period specific to each patient, and the potential delays induced by pacing versus intrinsic depolarization and conduction in a given patient [ 1 - 4 ]. As a result, the mean optimal AV intervals determined by us cannot be applied to other patient cohorts with the same basic disease. Individual programming of the AV interval is therefore necessary. Conclusion In summary, our findings confirm that Ritter's method can be reliably applied for patients with normal and with reduced left ventricular pump function. The only prerequisite is a continuous ventricular stimulation. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544593.xml |
554780 | Quality of sleep in patients with schizophrenia is associated with quality of life and coping | Background While sleep disturbance is widespread in schizophrenia it is less clear whether sleep disturbance is uniquely related to impaired coping and perceived quality of life. Methods We simultaneously assessed sleep quality, symptoms, and coping in 29 persons with schizophrenia or schizoaffective disorder in a post acute phase of illness. Assessment instruments included the Pittsburgh Sleep Quality Index; the Positive and Negative Symptom Scale; the Heinrichs Quality of Life Scale; and the Ways of Coping Scale. Multiple regressions were performed predicting quality of life and coping from sleep quality controlling for age and symptom severity. On a subset of seven subjects non-dominant wrist actigraphy was used as an objective check of their self-reported poor sleep. Results Analyses revealed that poor sleep quality predicted low quality of life (r = -0.493; p = .022) and reduced preference for employing positive reappraisal when facing a stressor (r = -0.0594; p = 0.0012). Actigraphy confirmed poor sleep quality in a subset of subjects. They had shorter sleep duration (p < .0005), shorter average sleep episodes (p < .005) and more episodes of long awakening (p < 0.05) than community norms. Conclusion The results are consistent with the hypotheses that poor sleep may play a unique role in sustaining poor quality of life and impaired coping in patients with schizophrenia. These associations may hold for community controls as well. | Background Many persons with schizophrenia report chronically disturbed sleep [ 1 , 2 ]. Independent of the phase of illness, sleep disturbance documented by polysomnography include: difficulties falling asleep, awakening too early and being unable to go back to sleep, a preference for being awake during the evening, reduced deep or slow-wave sleep (the most restorative stage of sleep), and short REM latencies [ 3 - 6 ]. In addition to being a source of distress, various forms of sleep disturbance have also been linked to heightened levels of thought disorder [ 7 ] and symptoms of excitement [ 8 ] and may portend relapse [ 9 ]. While sleep disturbance appears widespread in schizophrenia and is related to clinical features, less clear is whether it is also related to impaired coping that characterizes the disorder. Is poor sleep quality another factor that uniquely contributes to the difficulties persons with schizophrenia experience coping with stressors, and sustaining relationships and work? There appear to be several reasons to hypothesize that it may. Impaired sleep may make it difficult for persons with schizophrenia to cope with stressors [ 10 ]. Persons with schizophrenia and impaired sleep might feel, for instance, especially exhausted and highly inclined to avoid stressors and have difficulty seeing the positive aspects of daily challenges. Consistent with this, at least one study finds that damaged sleep quality in schizophrenia relates to longer periods of time spent in bed [ 2 ]. With low energy level and need for more time in bed, it seems a matter of intuition that persons might have great difficulty sustaining interpersonal relationships and adapting to the demands of a work setting. Additionally, beyond its effects on coping, we might expect persons with impoverished sleep, irrespective of their symptom level, to have less social and vocational satisfaction than those with better sleep. Indeed, one study indicates that sleep quality is associated with quality of life for patients with schizophrenia [ 11 ]. To examine relationships between sleep quality, coping and quality of life, we simultaneously assessed these domains along with symptom level in a group of patients with chronic schizophrenia and schizoffective disorder in a post acute phase of illness. To assess sleep quality we used the Pittsburgh Sleep Quality Index (PSI [ 12 ]), a standardized self-assessment tool that gauges sleep quality during the past month. We chose the PSI because it has been successfully used in several studies of persons with schizophrenia [ 2 , 11 , 13 , 14 ]. To assess coping we used the Ways of Coping Questionnaire (WCQ), a widely used instrument that measures the relative degree of preference for a number of coping strategies and which is sensitive to the maladaptive coping preferences common to schizophrenia [ 15 ]. To assess symptoms and quality of life, we used two instruments that are the gold standard in schizophrenia research: the Positive and Negative Syndrome Scale (PANSS [ 16 ]) and the Quality of Life Scale (QLS [ 17 ]). We predicted that, controlling for levels of positive and negative symptoms, poorer sleep quality would predict poorer quality of life overall, a greater preference for avoidant coping, and a lesser preference for coping by positive reappraisal. Some doubt the abilities of persons with schizophrenia to report their own sleep patterns accurately. However, a study comparing polysomnography with subjective measures of sleep found that they were highly correlated in a group of patients with chronic schizophrenia [ 18 ]. Similarly, others found that persons with severe mental illness can generally report the quality of their lives as well as those with other non-psychiatric illnesses [ 19 , 20 ]. In addition, we wanted to know if actigraphy is an effective way of demonstrating sleep problems in the post-acute phase of schizophrenia. To address this question, we used actigraphy to monitor sleep patterns in a subset of this patient population. Wrist actigraphy consists of monitoring locomotor activity with a motion sensor slightly bigger than a wrist-watch that is worn on the non-dominant wrist. It is a common method used to assess sleep [ 21 ]. Actigraphy is valid and reliable for evaluating sleep patterns in insomnia, in diagnosing circadian rhythm disorders, and in assessing sleep in subjects who are unlikely to tolerate polysomnography [ 22 ]. There are small studies that suggest the feasibility of using actigraphy in assessments of sleep disturbance in patients with schizophrenia [ 23 , 24 ]. Methods Participants All subjects met the Structured Clinical Interview for DSM IV (SCID [ 25 ]) diagnosis DSM-IV-TR [ 26 ] criteria for schizophrenia or schizoaffective disorder. All were in the post acute phase of illness; with no hospitalizations or medication changes for at least one month. Most were patients with chronic schizophrenia who were in a VA day treatment setting. All patient-subjects participated in an informed consent procedure and signed informed consent forms for this research. A tabulated summary of the participants' characteristics is in Table 1 . Table 1 Subject symptoms and demographics Subject Characteristics Mean (S.D.) Subjects Enrolled 29 Men 27 Women 2 Age 48 (7) Education 12 (2) Age at first hospitalization 25 (9) Number of hospitalizations 12 (6) SCID diagnosis Schizophrenia 23 Schizoaffective disorder 6 PANSS components Positive 19 (5) Negative 19 (6) Cognitive 19 (5) Emotional 13 (4) Excitement/Hostility 9 (3) Medication (CPZ equiv.) 755 (752) Instruments Actigraphy Sensors (Model # 24.000, Ambulatory Monitoring Inc, Ardsley, NY) slightly bigger than a wristwatch were worn by subjects on their non-dominant wrists for 21 days. Actigraphic logs of the non-dominant arm have a high correlation with gross locomotor activity [ 27 ]. Actigraphy is validated and reliable for evaluating sleep patterns [ 22 ]. The data from each actigraph was processed by the software included with the actigraphic monitors. Pittsburgh Sleep Quality Index (PSI) The PSI is an effective instrument used to measure the quality and patterns of sleep. It differentiates "poor" from "good" sleep by measuring seven subscales: Subjective Sleep Quality, Sleep Latency, Sleep Duration, Habitual Sleep Efficiency, Sleep Disturbances, Use of Sleeping Medication, and Daytime Dysfunction over the last month. The client self-rates each of these seven areas of sleep by answering nine questions. Scoring of answers is based on a zero to three scale, and a score of three reflects the negative extreme on the Likert Scale. A global sum of "5" or greater indicates a "poor" sleeper. The PSI has internal consistency and a reliability coefficient (Cronbach's alpha) of 0.83 for its seven components. Numerous studies using the PSI have supported high validity and reliability. Positive and Negative Syndrome Scale (PANSS) The PANSS is a 30 item rating scale completed by clinically-trained research staff at the conclusion of chart review and a semi-structured interview. For the purposes of this study the five PANSS factor analytically derived components are used: Positive, Negative, Cognitive, Excitement and Emotional discomfort [ 28 ]. Quality of Life Scale (QLS [ 29 ]) The tool is a 21 item scale completed by clinically trained staff after a semi-structured interview and chart review that assesses quality of life. Items are scored on a 7-point scale with higher ratings representing higher levels of satisfaction. Items tap a range of essential aspects of psychosocial interactions incorporating four subscales: Interpsychic Foundations; Occupational Functions; Commonplace Objects; and Activities and Interpersonal Relationships. For the purposes of this study we used the sum of all items as an index of overall quality of life. Interrater reliability for this instrument has been reported to range between 0.85 and 0.93 [ 15 ]. Ways of Coping Questionnaire (WCQ [ 30 ]) The WCQ is a self-report instrument that asks participants to call to mind a recent stressor and then rate how often they have used 66 different behaviours to cope with that stressor. Scale scores are additively derived from individual items and divided by a total score to provide a relative score. This relative score reflects participants' relative preferences among a set of discrete coping strategies. Relative scores are generally preferable because, among other things, they control for response bias. For the purposes of this study we calculated the relative scores for two subscales: "Escape Avoidance," and "Positive Reappraisal." Escape Avoidance describes wishful thinking and behavioural efforts to actively escape or avoid the problem and includes items such as: "I refused to believe it had happened" and "I wished the situation would go away or be over with." Positive Reappraisal describes efforts to create positive meaning by focusing on personal growth and includes items such as: "I changed or grew as a person in a good way" and "I found new faith." Internal consistency assessed using Cronbach's alpha have been reported to range from 0.61 to 0.79. Procedures Following informed consent participants were administered the PSI. PANSS, QLS, and WCQ were completed within the previous month for another study of the effects of personality on function in schizophrenia. Diagnosis was determined using the SCID. Ratings of sleep quality were elicited by a research assistant blind to the results of the PANSS, QLS and WCQ. We fitted a subset of patients with actigraphs that they wore for 21 days. Statistical analysis The statistical analyses were Pearson correlations and Mixed Models analyses in SAS ® software. We also repeated the correlations for patients with schizophrenia separate from the patients with schizoaffective disorder. Results Mean scores ± SD on the tests were: Quality of Life total 51.73 ± 16.16; Pittsburgh Sleep Quality Index 11.6 ± 4.57; Ways of Coping Questionnaire subscales: "Escape Avoidance," 0.138 ± 0.062 and "Positive Reappraisal" 0.125 ± 0.079. There was no correlation between QLS and "Escape Avoidance" scores, but a modest positive correlation between QLS and "Positive Reappraisal" (r = 0.421, p = 0.021). To examine the associations between sleep and quality of life we next performed partial correlations, using the PSI total score to predict QLS total score and QLS subscores with age and positive and negative component scores as covariates. Similarly, we used PSI subscores to predict QLS total score. Results revealed that poor sleep was related to poor quality of life (total PSI vs total QLS, r = -0.493; p = .022). The PSI accounted for 24% of the variance in QLS. Total PSI did not correlate with any of the QLS subscores. The PSI subscores: "Subjective Sleep Quality", "Sleep Duration", and "Sleep Disturbances" correlated with total QLS at p < 0.05 but none were significant after making the Bonferroni correction for multiple comparisons. To examine the associations between sleep and coping style we next performed partial correlations, using the PSI total score to predict WCQ Escape Avoidance, and WCQ Positive Reappraisal score with age and positive and negative component scores as covariates. Results revealed that poor sleep was related to a reduced preference for Positive Reappraisal (r = -0.594; p = 0.0012). Sleep quality was found to be unrelated to Escape Avoidance. The PSI accounted for over 37 % of the variance in coping. Patients with schizoaffective disorder had worse sleep as measured by the PSI than did patients with schizophrenia (13.7 ± 4.4 versus 9.7 ± 4.7; t-test, p = 0.03). The two groups did not differ in total scores of the QLS or WCQ. The correlations between total PSI and total QLS, and between total PSI and Positive Reappraisal for patients with schizophrenia were significant (r = -0.516; p = 0.039 and r = -0.645; p = 0.006 respectively). The same correlations were not significant for patients with schizoaffective disorder. Seven participants with schizophrenia accepted the activity monitors and were willing to wear them continually for up to 3 weeks. Seven patients completed actigraphic monitoring, and the actigraphic results are in Table 2 . Actigraphy verified that participants with schizophrenia had less overall sleep and more interrupted sleep than published community norms. Table 2 Sleep measures for subject subset and controls* Sleep Measure Means Patients Controls Units Participants 7 >200 Sleep Duration 1359 c 1440 min/d Sleep Proportion 25 33 % of d # of Long Awake Episodes 10.2 a 6.7 ea d Mean Sleep Episode 28 b 60 min Longest Sleep Episode 105 c 231 min *Controls data provided by Ambulatory Monitoring a = p < .05; b = p < .005; c = p < .0005 Discussion The results are consistent with the hypothesis that poor quality sleep that typically characterizes schizophrenia may have a powerful impact on both patients' perception of their quality of life and their ability to cope with stress. Independent of age and symptoms, poorer sleep quality predicted poorer quality of life and greater difficulties appraising stressors in a positive light. This may suggest that among the deficits accompanying schizophrenia, poor sleep is often underappreciated. Impaired sleep may erode the ability of schizophrenic patients to cope with the routine stress associated with work and interpersonal relationships. Furthermore, chronic sleep deprivation may contribute to anergy that impairs attendance and work performance. Since the found associations are independent of symptom level they may be trait variables. These findings are consistent with polysomnographic studies of sleep in schizophrenia which show that many sleep deficits that are not dependent on the acuity of illness [ 3 - 7 ]. The findings are also consistent with a prior study of sleep quality and quality of life in patient with schizophrenia [ 11 ]. We had an insufficient number of participants to confirm a correlation between PSI subscores and total Quality of Life. The negative relationship between complaints of poor sleep quality and Preference for coping by Positive Reappraisal remained significant when the confounding effects of symptom acuity and age were partialled from the correlation matrix. One interpretation is that chronically disturbed sleep may erode both the ability to find positive meaning and the desire to achieve personal growth. Given the correlative nature of the data analysis, however, it is not possible to infer causality. It may be that poor coping or poor quality of life lead to greater difficulties sleeping or that another unmeasured variable may account for the relationships. We plan future longitudinal studies that may uncover if sleep changes typically precede or follow changes in either coping or quality of life. There are other limitations to this study; most participants were male and in their 40s. Further experiments will be needed which include females and males in earlier phases of illness. The association of disturbed sleep and both quality of life and coping may hold for community controls as well. Conclusion These findings may have important clinical implications. If poor sleep quality is indeed a critical factor in quality of life and coping impairments in schizophrenia, clinicians will need to focus on and aggressively treat sleep problems. Specific treatments could include training in sleep hygiene with a focus on regular waking and sleep times, avoiding naps, morning bright light, evening melatonin, or other hypnotic agents. Improved sleep may lead to improved ability to cope with stress, and increased energy. These would improve the quality of life and coping in patients with schizophrenia. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JH participated in the design and coordination of the study and performed the statistical analysis and helped to draft the manuscript. PL conceived of the study and participated in its design and helped to draft the manuscript. AM participated in the design of the study and helped to draft the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554780.xml |
506788 | The expanding role of Tax in transcription | The viral transactivator of HTLV-I, Tax, has long been shown to target the earliest steps of transcription by forming quaternary complexes with sequence specific transcription factors and histone-modifying enzymes in the LTR of HTLV-I. However, a new study suggests that Tax preferentially transactivates the 21-bp repeats through CREB1 and not other bZIP proteins. The additional transactivation of Tax-responsive promoters subsequent to initiation is also presented. This result highlights a potentially novel role of Tax following TBP recruitment ( i.e. initiation) and may expand the mechanism of Tax transactivation in promoter clearance and transcriptional elongation. | Viruses have long been a source of key scientific discoveries. Historically, they have contributed to our knowledge of transcription, cell cycle, and apoptosis. To date activated transcription in higher eukaryotic cells with or without chromatin is a great area of active research and many researchers use viral activators, including herpes virus VP16, adenovirus E1A, HIV-1 Tat and HTLV-I Tax to not only understand viral, but also basic mechanisms related to host control of vital cellular machineries, including transcription. Eukaryotic transcription has five distinct phases, pre-initiation, initiation, promoter clearance, elongation and termination, and is a tightly regulated and coupled process [ 1 ]. Viral transactivators, such as Tax, have long been shown to target the earliest steps of transcription by forming quaternary complexes with sequence specific transcription factors and histone-modifying enzymes in the LTR of HTLV-I. These Tax-containing complexes allow for increased recruitment of TBP (TFIID), GTFs, and RNAP II within the core promoter region, leading to the synthesis of viral RNA. However, determination of those cellular factors important for enhanced transcriptional activity, as well as the full scope of Tax transactivation, is still not fully elucidated. In the report by Ching et al. [ 2 ] the authors directly compare which HTLV-I enhancer motif is preferred by Tax. Each enhancer element (21-bp, CRE, AP1, SP1, κB, or SRE) was placed in an identical TATAA-context to generate a minimal HTLV-I promoter. Previous studies had utilized various promoters (which contain additional DNA elements) to highlight a particular enhancer element necessary for Tax transactivation. Thus, this is the first study to directly compare these elements in an identical setting. In the presence of Tax, the 21-bp repeat (also known as the viral CRE elements or TxREs) was found to be most responsive (70-fold above basal levels). The 21-bp repeat was clearly preferred by Tax, since other enhancer elements were only stimulated 10-fold or less. Previously, several studies suggested that Tax activation of the 21-bp repeats may be mediated by ATF-4 [ 3 - 5 ]. It was shown that Tax was able to interact with ATF-4 bound to the 21-bp repeats, enhance the binding of ATF-4 to the enhancer, and recruit CREB binding protein (CBP) to the viral promoter [ 5 ]. Recently, CREB1 and ATF-4, in addition to ATF-1 and ATF-2, were found to be present in vivo on the 21-bp repeats (viral CRE elements) in HTLV-I infected cells through chromatin immunoprecipitation (ChIP) assays [ 6 ]. By using dominant negative mutants of CREB1, ATF-4 (CREB2/TAXREB67), Fos, and LZIP, Ching et al. demonstrated that among the various bZIP proteins, CREB1 was clearly favored for Tax transactivation of the 21-bp repeats. Additionally, CREB1 has also been found to primarily bind at the 5' LTR (rather than the 3' LTR) in vivo within HTLV-I infected cells, lending support to the idea that CREB1 is important for HTLV-I activated transcription [ 7 ]. If CREB1 is the dominant bZIP protein that is needed for Tax transactivation of the LTR, then what is the purpose of the additional bZIP proteins? Besides contributing to Tax transactivation, could these bZIP proteins help to exclude negative regulators from the LTR? A report by Basbous et al. [ 8 ] suggested that HBZ, which negatively down-regulated transcription from the HTLV-I LTR, heterodimerized with ATF-4 and subsequently this complex was no longer able to bind to the 21-bp repeats. Only over-expression of ATF-4 was found to reverse the negative effects of HBZ on Tax activity. However, additional studies are still needed to understand the respective contribution of CREB1 and other bZIP proteins, such as ATF-4, to Tax transactivation in the context of wildtype virus and stably integrated viral promoters (i.e. correctly assembled chromatinized DNA templates both in vitro and in vivo ). Lastly, Ching et al. presented the intriguing possibility of Tax enhancing transcription following transcription initiation. To determine whether Tax functioned solely to target TBP to the TATAA-element or if additional events subsequent to TBP (TFIID) recruitment were promoted by Tax, the authors constructed four independent reporters. Each promoter contained the minimal TATAA-element from HTLV-I, HIV-1, SV-40, or E1b promoters, two 21-bp repeats, and five copies of the Gal4-binding site. TBP was artificially targeted to the TATAA-element thru Gal4-TBP. The authors reasoned that if Tax functioned strictly to recruit TBP to the TATAA-element, then additional enhancement of transcription would not be observed when Tax and Gal4-TBP were present. Interestingly, only the Tax-responsive promoters, i.e. HTLV-I and HIV-1, were both synergistically stimulated by the addition of Tax and Gal4-TBP. These results suggest that Tax may control downstream transcription subsequent to the initiation phase. Other viral transactivators have been shown to have a role at initiation and downstream events, such as elongation. The most notable of these has been Tat, the viral transactivator of HIV-1. Without cellular stimulation and Tat expression, RNAP II transcriptional elongation was shown to be inefficient, producing only short transcripts [ 9 ]. One major contributing factor of Tat-dependent transactivation is the elongation factor, pTEFb. pTEFb, composed of cyclin T1 and cdk9, associates with Tat leading to increased phosphorylation at specific sites on the heptad repeats of the CTD of RNAP II and promoting elongation. Elongation is highly dependent on the status of RNAP II CTD, since dissociation/association of factors have been shown to be dependent on CTD serine 5/serine 2 phosphorylation [ 1 , 10 ]. Hyperphosphorylation of CTD at serine 5 is associated with promoter clearance/early elongation, whereby initiation factors are released and the 5'capping machinery subsequently recruited. During processive elongation, there is a switch in CTD phosphorylation to serine 2 phosphorylation resulting in the loss of the capping machinery and the association of splicing, elongation and chromatin remodeling factors. In the case of HTLV-I, Tax has been shown not to associate with a CTD kinase [ 11 ] and a dominant negative mutant of cdk9 (the catalytic subunit of pTEFb) was found to increase Tax transactivation of the HTLV-I promoter [ 12 ]. Therefore, there is the possibility that other kinase complexes (small vs. large pTEFb complex or other cdk kinases) may aid in increased Tax transactivation. In this context, HTLV-I infected cells contain increased levels of cyclin E/cdk2 kinase activity, through sequestration of cdk inhibitor, p21/waf1, by cyclin D 2 /cdk4 complexes [ 13 , 14 ]. This kinase complex was able to phosphorylate RNAP II CTD and antibodies against cyclin E co-immunoprecipitated only the phosphorylated form of RNAP II from HTLV-I infected cells. Thus, if only indirectly, Tax may increase kinase activity resulting in enhanced CTD phosphorylation for steps following initiation, such as promoter clearance and/or elongation. Processive elongation is highly dependent on remodeling of chromatin structure [ 1 , 10 ]. A study by Corey et al. [ 15 ] demonstrated that disruption of SWI/SNF recruitment by an activator resulted in lack of chromatin remodeling, transcription elongation, and production of full-length hsp70 mRNA. Tax has been shown to associate with BRG1 components of the ATP-dependent chromatin remodeling complex, SWI/SNF, and increase Tax transactivation [ 16 ]. Disruption of BRG1 by siRNA led to a decrease in Tax transactivation. Therefore, Tax may target SWI/SNF complexes downstream of RNAP II in order to prevent stalling of RNAP II. This raises a number of questions such as does Tax bind to an elongating RNAP II complex? Does Tax help to recruit elongation factors, such as TFIIS or TFIIF? Finally, it should be emphasized that each stage of transcription is not an independent process; coupling of the transcriptional and RNA processing machinery is thought to increase the rate and specificity of these enzymatic reactions [ 1 ]. As shown in Figure 1A , acetylation of nucleosomes and other transcription factors/coactivators promote an open complex structure and RNAP II holoenzyme assembly. Initiation by Tax is dependent on the recruitment of CBP/p300 and p/CAF by transcription factor/Tax complex at the 21-bp repeats (viral CRE elements). Phosphorylation of RNAP II CTD is important for loading of the 5' capping machinery to allow for rapid capping of nascent pre-mRNA, ensuring protection for the transcript from degradation. During promoter clearance (early elongation), site specific phosphorylation of the CTD is modified to allow for sequestration of splicing machinery and elongation factors, and release of the capping machinery. Assembly of SWI/SNF factors with Tax downstream of the elongation phase RNAP II complex remodels chromatin structure, promoting RNAP II processivity. Thus, the presence of Tax for initiation and possibly promoter clearance and/or elongation will help to increase viral transcription and mRNA processing overall (Figure 1B ). While the results by Ching et al. are preliminary at this time, Tax transactivation post-initiation is indeed a novel concept. Further detailed analysis of Tax at both the LTR of HTLV-I and downstream of this region will help to resolve many of these questions and provide important insight into the transcription field. Figure 1 Effect of Tax on transcription. A) Schematic representation of proximal promoter of HTLV-I. Tax binding to CBP/p300 with either p/CAF or bZIP transcription factors ( e.g. CREB1) leads to increased acetylation and interaction with the basal transcription machinery. Tax binding to SWI/SNF downstream of start site may help to remodel restrictive chromatin structure and aid in promoter clearance and elongation. B) The possible effect of Tax on gene expression network. The sequential steps of transcription (initiation, elongation, and termination) are intricately linked together and to mRNA processing and export (adapted from ref. 1). Thus, the effect of Tax on initiation and possibly elongation (both early promoter clearance and processive elongation events) would contribute, albeit indirectly, to RNA processing and export. Abbreviations HTLV-I, human T cell leukemia virus, type I CRE, cAMP response element CREB, cAMP response element binding protein ChIP, chromatin immunoprecipitation RNAP II, RNA polymerase II CTD, C-terminal domain HIV-1, human immunodeficiency virus, type 1 LTR, long terminal repeat TBP, TATA binding protein TxREs, Tax-responsive elements GTFs, general transcription factors TAR, transactivation region Competing Interests None declared. Authors' contributions Both authors contributed equally to the structure and content of the manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC506788.xml |
535342 | Platelet-derived NO slows thrombus growth on a collagen type III surface | Nitric oxide (NO) is a free radical that plays an important role in modulating platelet adhesion and aggregation. Platelets are a source of vascular NO, but since erythrocytes avidly scavenge NO, the functional significance of platelet-derived NO is not clear. Our purpose was to determine if NO from platelets affects platelet thrombus formation in the presence of anticoagulated whole blood in an in vitro parallel plate flow system. We studied platelet adhesion and aggregation on a collagen type III surface in the presence of physiologically relevant fluid mechanical shear stress. We found that certain receptor mediated agonists (insulin and isoproterenol) caused a concentration dependent reduction in thrombus formation at a shear rate of 1000 s -1 . This effect was mediated by NO since it was abolished in the presence of the NO inhibitor L-nitro-arginine-methyl-ester (L-NAME). As expected, at venous levels of shear rate (100 s -1 ) neither of the agonists had any effect on thrombus formation since platelet adhesion does not depend on activation at these low levels of shear. Interestingly, at a shear rate of 2000 s -1 the addition of L-NAME caused an increase in platelet coverage suggesting that shear, by itself, induces NO production by platelets. This is the first demonstration of shear stress causing platelets to produce an inhibitor of platelet activation. These results demonstrate that the development of a platelet thrombus is regulated in a complex way and that platelets produce functionally significant amounts of NO even in the presence of whole blood. | Introduction Platelet activation and aggregation play an important role in the development of cardiovascular disease, which is the leading cause of death in the United States. Over the last several years, enormous advances have been made in understanding the molecular mechanisms that regulate platelet function. A general paradigm has emerged that endothelial cells synthesize and release substances that inhibit platelet activation (e.g. nitric oxide and prostacyclin) except near a site of vascular injury, while platelets synthesize and release substances that promote further platelet activation. In contrast to this paradigm, several recent studies have suggested that, in the presence of certain agonists (IGF-1, adenosine diphosphate, insulin, and isoproterenol), platelets will produce nitric oxide (NO), a potent inhibitor of platelet activation [ 1 - 5 ]. Although these studies clearly showed that platelets produce NO, the physiological relevance of this NO is not clear since NO is highly reactive, has a short half-life, and the rate of NO production is difficult to quantify. Therefore, there is a need to determine if the NO derived from platelets is sufficient to modulate platelet function and alter thrombus growth in the presence of whole blood. Platelet-derived NO is synthesized by membrane-bound endothelial-type nitric oxide synthase (eNOS). A common signaling mechanism shared by many eNOS agonists is binding to a surface receptor, followed by activation of phosphatidylinositol-3-kinase (PI3K), which results in phosphorylation of eNOS at serine 1179 through Akt [ 6 - 8 ]. The activated eNOS converts L-arginine to L-citrulline and produces NO as a result [ 9 ]. Nitric oxide is a water soluble free radical that can bind to the heme-soluble site of guanylate cyclase in platelets and smooth muscle cells, which increases synthesis of cyclic guanosine monophosphate (cGMP) [ 5 , 10 ]. cGMP can bind to phosphodiesterase III (PDE III), which reduces metabolism of cyclic adenosine monophosphate (cAMP) [ 10 ]. Elevated levels of cGMP and cAMP can result in increased activity of protein kinase G (PKG) and protein kinase A (PKA), which inhibit protein kinase C (PKC) activation and intracellular Ca 2+ mobilization [ 5 ]. The consequence of this signal transduction cascade is the inhibition of platelet activation and the relaxation of vascular smooth muscle resulting in blood vessel dilation. As a small, hydrophilic free radical NO is highly diffusible in the aqueous environment of the blood. However, it is also highly reactive with a very short half-life estimated to be only on the order of a few seconds [ 11 ] in the blood. Previous studies that demonstrated NO production in platelets were done in the absence of erythrocytes [ 2 - 4 ]. It is still not clear if the amount of NO produced by platelets, estimated at about 5 × 10 -17 mole NO/platelet (determined by microelectrode in PRP for 2 minutes following addition of 5 μM ADP), is sufficient to alter platelet function [ 1 , 2 ]. The purpose of our study was to determine if platelet-derived NO plays a role in thrombus formation in the presence of shear stress. We examined the effect of several external factors on platelet thrombus formation, including insulin, the β-adrenoceptor agonist isoproterenol, and shear stress. Previous studies showed that insulin and isoproterenol both induced NO formation by platelets [ 3 , 4 ]. Shear stress is an important component of the environment of the platelets and can cause alterations in platelet function, although the effect of shear on NO synthesis in platelets is unknown [ 12 ]. Our aim was to show that platelet-derived NO plays a direct role in inhibiting thrombus formation to a vascular injury and can be stimulated by different external agonists through a common NO signaling pathway. Results Insulin and isoproterenol slow the growth rate of mural thrombi Platelets from whole blood adhered avidly to the collagen-coated surface in the presence of physiologically relevant levels of fluid mechanical shear stress. Very little, if any, platelets were detected on the albumin-coated portion of the slide. Detectable levels of platelet adhesion were evident within 20–30 seconds of the initiation of blood flow over the surface. Platelet adherence occurred predominantly at the interface between albumin and collagen and increased as a function of time similar to results that have been obtained by others using a similar system [ 13 - 16 ] Representative images of platelet accumulation on collagen at a shear rate of 1000 s -1 are shown in Figure 1 . In these images, the flow was from left to right. The albumin/collagen interface is clearly evident in the images from later time points. At other shear rates, platelet accumulation on the surface was also abundant as illustrated in Figure 2A . These results are in qualitative agreement with previously published results [ 13 ]. The slower rate of platelet deposition at higher levels of shear has been attributed [ 17 ] to the increased level of fluid mechanical drag on the platelets preventing them from forming stable attachments to the surface. Figure 1 Representative images of time-dependent platelet adhesion and aggregate formation on collagen type III. Single platelets and platelet aggregates adherent on the surface appear bright and were visualized using epi-fluorescence video microscopy. Platelets adhered abundantly on the collagen surface, particularly near the upstream interface between collagen and albumin. No adhesion was observed on the albumin-coated surface. Flow was from left to right and the shear rate was 1000 s -1 . These results are typical of 5 separate experiments. Figure 2 Dose dependence of thrombus formation. The extent of platelet adhesion onto the surface, as assessed by the percentage of the collagen-coated surface covered by platelets, is shown as a function of time. Perfusions for shear [100 (■), 1000 (□), 1500 (●), and 2000 (○) s -1 ] were performed for 5 minutes (Figure 2A). Perfusions for control (■), insulin [100 (◆) and 1000 pM (◇)] and isoproterenol [100 (▲) and 1000 μM (△)] were performed at 1000 s -1 (Figures 2B-C) for 6 minutes. The results presented are mean ± s.e.m. of at least 5 experiments using 5 different blood donors. The effect of insulin and isoproterenol on mural thrombus formation was studied and the results are presented in Figures 2B and 2C as the percent coverage of the collagen-coated surface as a function of time. Increasing the concentration of either insulin (0, 100, 1000 pM) or isoproterenol (0, 100, 1000 μM) at a shear rate of 1000 s -1 resulted in increasing inhibition of platelet accumulation on the surface. Higher concentrations of either insulin or isoproterenol had no additional affect on the extent of platelet accumulation on the surface (data not shown). Inhibition of thrombus formation was significant (p < 0.05) up to 6 minutes with 1000 pM insulin and up to 3 minutes with 1000 μM isoproterenol. Significance was determined with SPSS using the Post Hoc test as described in Methods. Agonist induced reduction in thrombus formation depends on NO and shear rate In order to investigate the mechanism of insulin and isoproterenol induced reduction in platelet accumulation on a collagen surface, we perfused blood through the flow chamber in the presence of agonist (either insulin or isoproterenol) as well as L-NAME, a specific inhibitor of nitric oxide production. The results are presented in Figures 3C,3D for the shear rate of 1000 s -1 . In the presence of L-NAME as well as either insulin or isoproterenol, thrombus formation is increased, returning to levels seen in the absence of added agonist. This suggests a role for nitric oxide in mediating the agonist induced reduction in platelet accumulation on the surface. Platelet thrombus formation on the surface at lower (100 s -1 ) and higher (2000 s -1 ) levels of shear rate are shown in Figures 3A,3B and 3E,3F respectively. At the lower, venous shear rate (100 s -1 ) platelet accumulation on the surface is not significantly altered by either insulin (500 pM) or isoproterenol (100 μM). Higher concentrations of either agonist had no affect as well (data not shown). In addition, L-NAME by itself or in combination with either agonist did not significantly affect platelet deposition on the surface (Figures 3A,3B ). At the higher, arterial shear rate of 2000 s -1 (Figures 3E,3F ), platelet accumulation was not significantly reduced by either insulin (500 pM) and isoproterenol (100 μM) as illustrated in Figures 3E,3F . Figure 3 Reduction in thrombus growth rate depends on platelet derived NO. Perfusions for control (■), insulin, 500 pM (◆), isoproterenol, 100 μM (▲), and samples preincubated with L-NAME [insulin (◇) and isoproterenol (△)] were performed at 100, 1000, and 2000 s -1 (Figures 3A-F). Results represent mean ± s.e.m. of at least five experiments with five separate blood donors. At the high shear rate of 2000 s -1 we observed a curious result when the blood was treated with L-NAME but in the absence of any agonist. The results are presented in Figure 4 . We found that in the presence of L-NAME, thrombus formation increased either with (data not shown) or without agonist present (Figure 4C ). This result suggests a role for nitric oxide in modulating the rate of platelet deposition onto a collagen surface at a shear rate of 2000 s -1 even in the absence of insulin or isoproterenol. Figure 4 Shear stress causes a slowing of thrombus growth rate that depends of platelet derived NO. Platelet coverage values for each study are the mean ± s.e.m. of 5 separate experiments, each using a different donor. Perfusions for control (■) and L-NAME (□) samples were conducted at 100, 1000, and 2000 s -1 (Figures 4A-C) for 6–10 minutes. Effect of insulin and isoproterenol on guanylate cyclase activity We further investigated the mechanism of platelet derived nitric oxide inhibition of thrombus formation by using the guanylate cyclase inhibitor 1H-[ 1 , 2 , 4 ]oxadiazolo [4, 3-a]quinozalin-1-one (ODQ), since the primary target of NO in platelets is guanylate cyclase [ 18 , 19 ]. As illustrated in Figure 5A , thrombus formation at 1000 s -1 and in the presence of insulin was slightly increased by addition of ODQ. No significant effect of ODQ on thrombus formation was observed with isoproterenol (Figure 5B ) or in the absence of agonist (Figure 5C ). This is not surprising since our other studies at this shear with isoproterenol (Figure 2D ) or in the absence of agonist (Figure 3B ) pointed out that the effect on platelet thrombus formation was also very small. Interestingly, we see that ODQ causes an increase in thrombus formation at 2000 s -1 if either insulin, isoproterenol, or no agonist is used (Figures 5D,5E,5F ). This suggests that at 2000 s -1 , guanylate cyclase activation is occurring even in the absence of added external agonists. Figure 5 Effect of guanylate cyclase on platelet adhesion kinetics. Platelet coverage values for each study are the mean ± s.e.m of 4 separate experiments, each using a different donor. Perfusions were conducted at 1000 and 2000 s -1 (Figures 5A and 5B) for 6 minutes with insulin, 500 pM (■), isoproterenol, 1000 μM (▲) and samples preincubated with guanylate cyclase inhibitor ODQ [insulin (□) and isoproterenol (△)]. Discussion The purpose of this study was to determine if nitric oxide from platelets affects platelet function. Previous studies have identified the vascular endothelium as the primary source of NO in the blood. Only recently have platelets been identified as an additional source of NO. The role of platelet derived NO in modulating platelet function has not been established. Most of the other studies that have examined NO production by platelets were performed with platelet rich plasma or with washed platelets. This was required in order to increase the platelet concentration to the point where NO levels could be assessed. However, since these studies were performed in the absence of erythrocytes, it is difficult to extrapolate the results to the in vivo situation where hemoglobin in red cells efficiently scavenges the nitric oxide free radical. Therefore, the experiments in our current study were designed to measure platelet function in a vascular injury model where the platelets remain in whole blood. Several recent studies have demonstrated that platelets produce NO in response to agonists including insulin, isoproterenol, and ADP [ 2 , 4 ]. Using an in vitro parallel plate flow chamber, we show in this study that the rate of mural thrombus growth on a collagen III surface is modulated by platelet derived NO. This is the first study to show that platelet derived NO can alter platelet function in whole blood. We found that insulin, isoproterenol, and shear stress could, under the right circumstances, modulate platelet function through an NO-mediated mechanism. In fact, we found that at a shear rate of 2000 s -1 , but not at either 100 or 1000 s -1 , platelet adhesion to the surface was increased in the presence of L-NAME, an inhibitor of nitric oxide production. This result suggests that platelets produce nitric oxide at higher shear rates but not at lower levels of fluid mechanical stress. This result is consistent with the observation that elevated levels of shear stress can cause platelet activation [ 20 , 21 ] and that nitric oxide production may be a common feature of platelet activation regardless how activation is initiated [ 8 , 22 ]. At the low, venous level of shear (100 s -1 ) that we tested, platelet derived NO did not appear to play a role in modulating platelet accumulation on the surface. This result was not surprising due to the many types of adhesion receptors on platelets that function well in a low shear environment. The primary platelet receptors for collagen III on a surface are GPVI and α 2 β 1 [ 23 , 24 ]. Additionally, the GPIb-V-X receptor complex can mediate platelet adhesion through interactions with soluble and surface associated von Willebrand Factor [ 25 ]. Because of the interaction with collagen, the platelets become activated. Activation induces an extensive series of intracellular signaling events to take place, resulting in the conversion of the integrins α 2 β 1 and α IIb β 3 into a high affinity state through a process termed inside-out signaling. In this high affinity state, these integrins are capable of supporting platelet-platelet interactions and promoting thrombus growth. Therefore, platelet adhesion at low levels of shear rate can be mediated by a number of different adhesion molecules, some of which do not require platelet activation. Our results are consistent with this model since we found that platelet derived nitric oxide did not inhibit the rate of thrombus growth at these low, venous levels of shear rate. At intermediate shear (1000 s -1 ), integrins α 2 β 1 and α IIb β 3 play a more significant role in platelet adhesion and recruitment. These platelet integrins are essential for thrombus formation at intermediate to high shear [ 26 ]. Furthermore, these integrins must become activated, that is convert into a high affinity conformation, in order for them to be capable of supporting adhesion at these high levels of shear [ 27 ]. This requires intracellular signal transduction events and platelet activation, a process that is inhibited by nitric oxide. Our results (Figures 1A and 1B ) suggest that insulin and, to a lesser extent, isoproterenol can both cause a decrease in the rate of growth of thrombi and that this decrease is concentration dependent. Furthermore, we showed that the mechanism of this inhibition of thrombus growth involves a nitric oxide/guanylyate cyclase pathway (Figures 2B and 4A ). Our results with the inhibitors of platelet signaling, L-NAME and ODQ, are consistent with previous studies. L-NAME completely abolished the anti-thrombotic effects of both insulin and isoproterenol at a shear rate of 1000 s -1 showing that these agonists act through a pathway that involves nitric oxide. ODQ was effective in inhibiting the action of insulin, but was less effective in inhibiting the action of isoproerenol. This result is consistent with earlier findings that suggested that isoproterenol inhibited platelet activation partially through a mechanism involving adenylate cyclase [ 28 ] rather than guanylyate cyclase. The results we present with the inhibitors L-NAME and ODQ at the highest shear rate, 2000 s -1 (Figures 2C , 4C , and 4D ) failed to demonstrate a role for platelet-derived NO or signaling through guanylyate cyclase. However, in the absence of added agonist, shear stress, by itself was able to induce platelets to produce NO (Figures 3C and 5B ). Although there has been no previous evidence for shear-dependent NO production in platelets, both in vitro and in vivo studies have demonstrated shear stress upregulates eNOS activity in endothelial cells [ 7 , 29 ]. The mechanism is not completely understood but involves cytoskeletal proteins and the activation of a series of kinases including PI3 kinase and Akt as well as Hsp90 [ 30 , 31 ]. A similar pathway for the activation of eNOS has been recently characterized in platelets in response to insulin [ 8 ]. Conclusions Our study focused on quantifying the effect of platelet-derived NO on platelet adhesion and aggregation on a surface in response to external factors (i.e. insulin, isoproterenol, and shear) and in the presence of whole blood. Platelet-derived NO did not affect platelet adhesion at low shear but had a significant effect at intermediate and high shear. Production of NO in platelets was dominated by receptor-mediated interactions at intermediate shear and mechano-transduction at high shear. This study and future work may lead to a better understanding of platelet-derived NO and its role in healthy and diabetic vascular wound healing. Methods Materials Low molecular weight heparin, HEPES, NaCl, collagen III from calf skin, bovine serum albumin (BSA), L-nitro-amine-methyl-ester (L-NAME), insulin from porcine pancreas, and isoproterenol were obtained from Sigma. Recombinant hirudin (r-hirudin) was obtained from Pentapharm and 1H-[ 1 , 2 , 4 ]oxadiazolo [4, 3-a]quinoxalin-1-one (ODQ) was obtained from Cayman Chemical. Mepacrine (quinacrine) was obtained from ICN Biomedicals. Fluorescein isothiocyanate (FITC) was generously donated by Paul Friese, University of Oklahoma Health Sciences Center, Oklahoma City, OK. The S12 anti-P-selectin monoclonal antibody was generously donated by Roger P. McEver, Oklahoma Medical Research Foundation, Oklahoma City, OK. Glass cover slips (24 × 50 mm) were obtained from Fisher Scientific. Preparation of Glass Coverslips Each cover slip was washed with 20 mL nanopure H 2 O and 10 mL 95% ethanol. After washing was complete, the cover slips were then placed in a 95% ethanol bath for at least 12 hours. Before use, each cover slip was rinsed with an additional 20 mL 95% ethanol and allowed to air-dry for 15 minutes. Protein coating of Cover slips Collagen III solution was prepared at 0.8 mg/mL in 17 mM acetic acid (pH 2.6) at least 24 hours prior to use. BSA solution was prepared at 0.1% in 10 mM HEPES/115 mM NaCl buffer (pH 7.4). Half of each cover slip was coated with collagen and allowed to incubate for 4 hours in a humidified environment (80–90%) at room temperature (~24°C). After incubation, each slide was rinsed with 10–15 mL of HEPES/NaCl buffer solution to remove excess collagen and coated with 0.1% BSA solution for at least 1 hour. Platelet Preparation Venous blood collected from healthy donors (30–60 mL, depending on shear rate and length of experiment) was anticoagulated with low molecular weight heparin to a final concentration of 20 U/mL or r-hirudin to a final concentration of 40 anti-thrombin units/mL (ATU/mL). The fluorescent dye mepacrine was then added to a final concentration of 10 μM and allowed to incubate for 10–15 minutes. All donors gave informed consent to participate in our study according to methods approved by the University of Oklahoma Institutional Review Board. Flow Experiments The arterial flow environment was modeled with an in vitro parallel plate flow chamber similar to those previously characterized [ 32 , 33 ]. The dimensions of the flow channel were the 0.013 × 1.3 cm for experiments done at a shear rate of 100 s -1 , 0.013 × .8 cm for shear rate of 500 s -1 , and 0.013 × .25 cm for shear rates of 1,000 and 2,000 s -1 . In some studies, 500 pM insulin or 100 μM isoproterenol was added to the anti-coagulated blood 10 minutes before the start of an experiment. In other studies, L-NAME, an inhibitor of platelet NO synthesis was added at a concentration of 200 μM for 20 minutes before the start of an experiment. In additional studies, ODQ, an inhibitor of guanylate cyclase was added at a concentration of 100 μM for 20 minutes before the start of an experiment [ 18 , 19 , 34 ]. In still other studies, combinations of L-NAME or ODQ and insulin or isoproterenol were added as described above, except that L-NAME or ODQ was added 20 minutes prior to insulin or isoproterenol addition. In each experiment, blood was perfused for 5 – 10 minutes. In all studies, blood was used in experiments within two hours of collection. The order of experiments was varied randomly to insure that time-dependent platelet phenotype was not skewing the results. Additionally, flow cytometric studies of platelets showed no significant change in platelet activation, as measured by P-selectin expression, over the course of 4 hours (data not shown). Microscopy and Imaging Systems A syringe pump provided the flow through the flow chamber. The flow chamber was placed on the stage of a Nikon Diaphot 300 inverted microscope. The illumination was provided by a 75W Xenon light source passing through a 480 nm excitation filter and a 40 × fluorite objective lens. The fluorescent emission from adherent platelets was passed through a 514 nm filter and converted to analog signal by an image intensifier and CCD camera. The image was recorded on VHS tape for subsequent analysis. The extent of mural thrombus formation on the surface was quantified in terms of the percent coverage of a representative area of the collagen-coated surface at the interface between the collagen and albumin. The analyzed area was approximately 100 μm wide by 150 μm in the direction of flow. Digital image analysis was performed on an SGI Indy workstation running the ISEE ® image analysis software by Inovision. A background image was acquired after blood began flowing over the surface but before adhesion of any platelets. This image was subtracted from subsequent images. Platelets were identified based on size and intensity using adjustable parameters. Images were acquired and analyzed every 10–30 seconds over the course of each experiment, which lasted from 5–10 minutes. Statistical Analysis Statistical significance of the results was assessed with the aid of the software package SPSS (version 11.5 for Windows). The statistical tests used were the Post Hoc test for multiple comparisons and the student's t-test. Results were deemed significant if p < 0.05. Abbreviations BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; eNOS, endothelial nitric oxide synthase, FITC, fluorescein isothiocyanate; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; L-NAME, L-nitro-amine-methyl-ester; NO, nitric oxide; ODQ, [ 1 , 2 , 4 ]oxadiazolo [4, 3-a]quinoxalin-1-one; PI3K, phosphatidylinositol-3-kinase; PRP, platelet rich plasma; vWF, von Willebrand factor Competing Interests The authors declare that they have no competing interests. Authors' contributions RW performed all of the experiments and MN conceived of the project and coordinated the data analysis. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535342.xml |
524501 | The pivotal role of cholesterol absorption inhibitors in the management of dyslipidemia | Elevated low-density lipoprotein (LDL)-cholesterol is associated with a significantly increased risk of coronary heart disease. Ezetimibe is the first member of a new class of selective cholesterol absorption inhibitors. It impairs the intestinal reabsorption of both dietary and hepatically excreted biliary cholesterol. Ezetimibe is an effective and safe agent for lowering LDL-C and non HDL-C. Short term clinical trials have established the role of ezetimibe monotherapy and its use in combination with statins. Furthermore, ezetimibe and statin combination therapy increased the percentage of patients who achieved their LDL-C treatment goal. Studies using surrogate markers of atherosclerosis have suggested a possible role of ezetimibe in combating atherosclerosis. Ezetimibe provides an effective therapeutic strategy for the management of homozygous familial hypercholesterolemia (HoFH) and sitosterolemia. The lack of outcomes and long term safety data is attributed to the relatively recent introduction of this medication. | Background Over 60 million Americans suffer from cardiovascular disease (CHD). The incidence of CHD and stroke has been on the rise partly because of the increase in life expectancy and the explosive epidemic of diabetes and the metabolic syndrome [ 1 ]. CHD is responsible for about 38% of the overall mortality in the United States making it the number one killer of Americans [ 2 ]. Animal and human studies have established the role of cholesterol in the development and progression of atherosclerosis. LDL-cholesterol (LDL-C) constitutes approximately 60–70 % of total serum cholesterol. Epidemiological studies directly implicated LDL-C to the development of atherosclerosis and CHD. Furthermore, LDL-C level appears to be directly related to the development and recurrence of CHD [ 3 ]. Animal studies suggested a protective effect of low LDL-C against atherosclerosis [ 2 ]. Multiple human trials examining the relationship of LDL-C lowering in primary and secondary prevention of CHD have demonstrated the impact of reducing LDL-C levels on decreasing CHD and CHD related mortality [ 4 - 8 ]. Most of the landmark CHD prevention trials involved the use of statin medications. LDL-C remains the primary target of treatment in most instances, and statins are the mainstay of LDL-C lowering treatment [ 9 ]. The National Cholesterol Education Program/ Adult Treatment Panel III (NCEP/ATP III) updated guidelines (table 1 ) for detection and treatment of dyslipidemia envisioned LDL-C below 100 mg/dL to be optimal for all patient risk categories. These more aggressive guidelines resulted in doubling of the number of patients that are not at target LDL-C levels as compared to previous guidelines [ 2 ]. Recent NCEP/ATP III update data suggested even lower LDL-C levels than previously advocated, making it harder to achieve the treatment in many instances and recommended the use of combination therapy if needed to help achieve the treatment targets. The NCEP/ATP III update emphasized "the lower, the better" hypothesis [ 10 ]. Table 1 Synopsis of the updated ATP III LDL-C Goals and Cut-points for TLC and Drug Therapy in Different Risk Categories and Proposed Modifications Based on Recent Clinical Trial Evidence Risk Category Goal TLC Drug Therapy High risk : CHD or CHD risk equivalents (10-year risk >20%) < 100 mg/dL (optional goal: <70 mg/dL) ≥ 100 mg/dL ≥ 100 (<100 mg/dL: consider drug options) Moderately high risk : 2+ risk factors (10-year risk 10% to 20%) < 130 mg/dL ≥ 130 mg/dL ≥ 130 mg/dL(100–129 mg/dL; consider drug options) Moderate risk : 2+ risk factors (10-year risk <10%) < 130 mg/dL ≥ 130 mg/dL ≥ 160 mg/dL Lower risk : 0–1 risk factor < 160 mg/dL ≥ 160 mg/dL ≥ 190 mg/dL (160–189 mg/dL: LDL-lowering drug optional) Cholesterol Absorption inhibitors Ezetimibe, a cholesterol absorption inhibitor, is the first agent of a new class of lipid-lowering compounds that selectively inhibits the intestinal absorption of cholesterol and related phytosterols. Ezetimibe undergoes extensive glucuronidation to an active metabolite in the intestinal mucosa [ 11 ]. Ezetimibe acts on brush border of the small intestine and decreases biliary and dietary cholesterol from the small intestine uptake into the enterocytes. Ezetimibe is primarily metabolized in the small intestine and liver via glucuronide conjugation with subsequent biliary and renal excretion [ 12 ]. Ezetimibe does not affect the absorption of fat-soluble vitamins, triglycerides, or bile acids [ 13 ]. After oral administration, ezetimibe is absorbed and extensively conjugated to a pharmacologically active phenolic glucuronide (ezetimibe-glucuronide) [ 14 ], the drug and its metabolite have a half-life of approximately 22 hours [ 8 ]. Concomitant food administration (high fat or non-fat meals) had no effect on the extent of absorption of ezetimibe when administered in the 10-mg clinical dose [ 15 ]. Ezetimibe and ezetimibe-glucuronide are highly bound (>90%) to human plasma proteins [ 16 ]. Plasma concentrations for total ezetimibe were about 2-fold higher in older individuals (>65 years), levels were similar in adolescents to healthy adults and may be higher in women than in men [ 8 ]. In patients with severe renal disease, ezetimibe level was increased approximately 1.5-fold, compared to healthy controls [ 17 ]. Ezetimibe had no significant effects on the bioavailability of warfarin, fenofibrate, HMG CoA reductase inhibitors, or digoxin [ 16 , 18 - 20 ]. Adverse experiences were reported in approximately 2% of patients treated with ezetimibe and included fatigue, arthralgia, diarrhea, abdominal pain and back pain. Angioedema and rash were reported after general clinical use of this medication [ 16 ]. With co-administration of ezetimibe and statins the adverse event profile was similar to that for statins alone. In a recently published case report, the authors described two patients whose creatinine kinase (CK) increased after the addition of ezetimibe to statin therapy causing one of the patients to experience myalgia and tendinopathy. This finding raises the question of whether ezetimibe can be implicated in precipitating increased risk of stain-associated myopathy [ 21 ]. The Role of Ezetimibe in Clinical Practice Indications for use 1. Primary Hypercholesterolemia (heterozygous familial and non-familial), Ezetimibe is indicated in this case for use as both mono and combination therapy. 2. The reduction of elevated total-C and LDL-C levels in patients with homozygous familial hypercholesterolemia (HoFH) either as primary or as an adjunct to other lipid-lowering treatments. 3. In patients suffering from Homozygous Sitosterolemia, as adjunctive therapy to diet for the reduction of elevated sitosterol and campesterol levels in patients with homozygous familial sitosterolemia. Monotherapy Multiple studies conducted to examine the effects of ezetimibe monotherapy have concluded that this drug was effective in lowering LDL-C versus placebo. Analysis of multicenter, double-blind, placebo-controlled trials demonstrated that ezetimibe at the 10 mg once daily clinically approved dose significantly modified cholesterol and cholesterol subtypes in patients with hypercholesterolemia when compared to placebo. Ezetimibe significantly lowered total-Cholesterol (TC) (12 %), LDL-C (18 %), apolipoprotein B (Apo B) (15 %), and triglycerides (TG) (7%) and increased high density lipoprotein (HDL-C) (3.5%) [ 22 - 24 ]. Lipoprotein (a) [Lp (a)] was not significantly affected by Ezetimibe 10 mg once a day treatment [ 25 ]. In a case series report, we analyzed the effects of Ezetimibe on cholesterol particle size and number using NMR technology (Lipo science, Raleigh, NC). We found that Ezetimibe lowered cholesterol particle number by a mean 26 % and had no significant effect on cholesterol particle size [ 26 ]. The effects of ezetimibe on cholesterol and its subtypes were not influenced by risk-factor status, gender, age, race, time of administration, or baseline lipid profile [ 22 ]. The overall incidence of adverse effects with ezetimibe monotherapy was similar to placebo. Combination therapy The struggle to achieve the NCEP/ATP III guidelines LDL-C goals through primary utilization of statins is often frustrating for the clinician. In a study to examine the efficacy of statin titration on attainment of LDL-C goal, the authors concluded that for high risk patients, approximately half were able to achieve their LDL-C goal at the appropriate statin starting dose, and only one third of the titration group were able to achieve the NCEP/ATP III cholesterol goal [ 27 ]. Now with the very recent publication of the update to the NCEP/ATP III, clinicians are faced with even lower goals of LDL-C, Making combination therapy a must in more cases than previously advocated by the NCEP/ATP III [ 10 ]. HMG-CoA reductase inhibitors (statins) act on the rate-limiting step to inhibit HMG-CoA conversion to mevalonate, effectively decreasing LDL-C synthesis. They result in a decrease of LDL-C ranging between 30–60 %, depending on the individual statin and the dose administered. Statin induced LDL-C lowering appears to be effective in reducing CHD and CHD related mortality and morbidity. The extent of CHD and CHD related events reduction is proportionate to the extent of LDL-C reduction [ 10 , 28 ]. LDL-C reduction trials have demonstrated a reduction in CHD related events by approximately 20–40% [ 4 - 8 ]. Reasons to initiate combination therapy to treat hyperlipidemia include: further LDL-C lowering, reducing side effects related to higher doses of statins, modifying other risk factors besides LDL-C such as HDL-C and TG. Increasing the dose of statins has a limited effect on reducing LDL-C, as it is well established that doubling the dose of a statins leads to a 5 % more reduction in total TC and 7 % more reduction in LDL-C with each doubling [ 29 ]. Although statins have demonstrated similarity in CHD related events, they are heterogeneous not only in LDL-C lowering efficacy but also in their safety profiles. The bulk of the statins effect on LDL-C occurs at the initial recommended dose and they are safer when used at doses below the maximal recommended dose. Statins are the most effective drugs known to modify LDL-C, but in terms of HDL-C and TG modifying capacity, other classes of lipid lowering medications used alone or in combination with statins offer higher efficacy. The metabolism of cholesterol is an intricate process that involves both produced and ingested cholesterol. The mechanism of action of HMG-CoA reductase inhibitors affects the production of cholesterol, whereas that of cholesterol absorption inhibitors affects absorbed cholesterol, thereby offering potential synergism of action when the medication are used in combination. Trials examining the efficacy have demonstrated synergism and consistency in LDL-C lowering in the absence pharmacokinetic interaction between the statins and ezetimibe. In a relatively large, multicenter study, involving patients with primary hypercholesterolemia already receiving statin monotherapy (but who had not met their NCEP ATP II target LDL-C goal), patients were randomized to receive either ezetimibe or placebo in addition to their current statin therapy. At the conclusion of this 8 week study, the ezetimibe and statin groups were found to have a significantly lower total-C, LDL-C, Apo B, and TG, and increased HDL-C when compared to the statin only and placebo groups. Furthermore, LDL-C reductions induced by ezetimibe were generally consistent across all statins groups [ 30 ]. Another multicenter, double-blind, randomized trial examined the effects of ezetimibe on patients suffering from (HoFH). At the initiation of the trial patients were receiving either atorvastatin or simvastatin. The addition of ezetimibe reduced LDL-C by an additional 20.5 % in contrast to only 6.7 % reduction that resulted from doubling the statin dose [ 31 ]. Similar results were demonstrated in high-risk patients with familial heterozygous hypercholesterolemia (HeFH) [ 32 ]. The addition of ezetimibe to statins is superior to treatment with statins alone in lowering non-HDL-C, ezetimibe co-administered with simvastatin lowered non-HDL-C by 47.1% whereas, simvastatin monotherapy lowered non-HDL-C by 33.6% when results were pooled across different doses [ 33 ]. In terms of modifying risk factors other than LDL-C, the co-administration of ezetimibe with statins had a more favorable effect on HDL-C and TG when compared to statins therapy alone [ 33 ]. In conclusion, the addition of ezetimibe to statins produced further lowering of LDL-C of approximately 15–20 % with no apparent increase in side effects. This effect was superior to that observed by doubling the dose of the statins. Furthermore, the lowering produced was consistent. Ezetimibe co-administration with fibric acid derivatives was examined in a randomized, evaluator-blind, placebo-controlled, parallel-group study of 32 healthy hypereholesterolemics. Ezetimibe co-administration with fenofibrate was found to produce clinically significant reductions in LDL-C (36.3%) compared to the fenofibrate group (22.3%) with a more favorable TG and HDL-C profile [ 34 ]. Sitosterolemia Sitosterolemia is a rare inherited disorder caused by mutation in either the ABCG5 or ABCG8 genes located on chromosome (2p21) [ 35 ]. First described by Bhattacharyya and Connor in 1974 in two sisters of German and German-Swiss ancestry with normal mental development. The patients presented with tendinous and tuberous xanthoma and elevation of beta-sitosterol, campesterol and stigmasterol (plant sterols) in the blood [ 36 ]. Affected individuals have increased intestinal absorption of plant sterols (mainly sitosterol) that are usually absorbed in minute amounts in normal individuals. Additionally, these patients have diminished clearance of plant sterols, leading to very high levels of plant sterols in the plasma. Patients suffering from sitosterolemia have severely depressed hepatic cholesterol biosynthesis, and decreased levels of HMG-CoA reductase enzyme [ 37 ]. Clinical manifestations include: tendon and tuberous xanthomas, episodes of hemolysis, accelerated atherosclerosis, and premature coronary artery disease. It is important to note that close to 50 % of these patients have normal cholesterol levels [ 38 - 41 ]. A recently reported trial demonstrated that treatment with ezetimibe reduces plant sterol levels in patients with sitosterolemia. The authors reported a decrease in sitosterol concentrations by 21% and campesterol by 24 % [ 42 ]. Ezetimibe and atherosclerosis It is generally accepted that atherosclerosis is an inflammatory disorder. It is believed that the atherosclerotic process begins with endothelial cell activation, which is triggered by multiple factors such as oxidized lipoproteins. Cholesterol lowering agents as a group have demonstrated great efficacy in prevention and cessation of the progression of atherosclerosis. The efficacy of ezetimibe in monotherapy or in combination on CHD morbidity and mortality has not been well established. One of the unique features of cholesterol absorption inhibitors is their ability to modify post-prandial hyperlipidemia. There is increasing evidence that post-prandial lipoproteins (particularly cholesterol-rich chylomicron remnant) are atherogenic. Ezetimibe has the potential to reduce the cholesterol content of chylomicrons by up to 60% [ 44 ], which may lead to a lower atherogenic potential of chylomicron remnants [ 43 ]. High-sensitivity C-reactive protein (hs-CRP) is an inflammatory mediator whose levels correlate with increased coronary risk. Ezetimibe co-administered with simvastatin resulted in significant incremental decreases in hs-CRP in patients with primary hypercholesterolemia. Changes in individual lipid parameters did not explain the observed decreases in hs-CRP and were possibly consistent with an additional anti-inflammatory effect compared with simvastatin monotherapy [ 45 ]. In a prospective trial to study effects of ezetimibe co-administered with atorvastatin in patients with primary hypercholesterolemia, ezetimibe plus atorvastatin significantly provided an additional (10%) lowering of hs-CRP versus atorvastatin alone [ 46 ]. The unanswered questions Ezetimibe and its class of cholesterol absorption inhibitors are new, and there is a lack of outcomes data to explore whether its cholesterol modifying effects will translate to lower CHD mortality and morbidity. The safety of this medication has not yet been established with long term trials data as most of the studies conducted were short term. With the advent and increased utilization of combination therapy in the management of dyslipidemia, further trials are needed to explore the efficacy, indications and safety profile of ezetimibe use in combination with Peroxisome proliferator-activated receptors (PPARs), niacin and bile acid resins. The increased popularity of special weight loss diets such as the high protein diet, poses questions of whether such diets will alter the efficacy or safety of cholesterol absorption inhibitors. Finally, the efficacy of statins in reducing CHD related events has lead to the controversial hypothesis regarding whether or not statins poses a pleiotropic (non lipoprotein) effect. If a pleiotropic effect exists, one might argue that a statin at a higher dose might be more beneficial than combination therapy producing the same effect. Conclusions Ezetimibe is the first clinically approved cholesterol absorption inhibitor. It is effective in lowering LDL-C as monotherapy or in combination with statins. The use of combination LDL-C lowering medication is expected to become a much more common modality of treatment, especially after the recent NCEP/ATP III update. Ezetimibe offers further lowering of LDL-C and non HDL-C that is consistent and probably safer than increasing the dose of the individual statin. It also provides another effective treatment option for HoFH and sitosterolemia patients. Because of its recent introduction, we still lack both outcomes and long term safety data. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524501.xml |
539236 | Neurotrophin and Trk expression by cells of the human lamina cribrosa following oxygen-glucose deprivation | Background Ischemia within the optic nerve head (ONH) may contribute to retinal ganglion cell (RGC) loss in primary open angle glaucoma (POAG). Ischemia has been reported to increase neurotrophin and high affinity Trk receptor expression by CNS neurons and glial cells. We have previously demonstrated neurotrophin and Trk expression within the lamina cribrosa (LC) region of the ONH. To determine if ischemia alters neurotrophin and Trk protein expression in cells from the human LC, cultured LC cells and ONH astrocytes were exposed to 48 hours of oxygen-glucose deprivation (OGD). Also cells were exposed to 48 hours of OGD followed by 24 hours of recovery in normal growth conditions. Cell number, neurotrophin and Trk receptor protein expression, neurotrophin secretion, and Trk receptor activation were examined. Results Cell number was estimated using an assay for cell metabolism following 24, 48 and 72 hours of OGD. A statistically significant decrease in LC and ONH astrocyte cell number did not occur until 72 hours of OGD, therefore cellular protein and conditioned media were collected at 48 hours OGD. Protein expression of NGF, BDNF and NT-3 by LC cells and ONH astrocytes increased following OGD, as did NGF secretion. Recovery from OGD increased BDNF protein expression in LC cells. In ONH astrocytes, recovery from OGD increased NGF protein expression, and decreased BDNF secretion. Trk A expression and activation in LC cells was increased following OGD while expression and activation of all other Trk receptors was decreased. A similar increase in Trk A expression and activation was observed in ONH astrocytes following recovery from OGD. Conclusions In vitro conditions that mimic ischemia increase the expression and secretion of neurotrophins by cells from the ONH. Increased Trk A expression and activation in LC cells following OGD and in ONH astrocytes following recovery from OGD suggest autocrine/paracrine neurotrophin signaling could be a response to ONH ischemia in POAG. Also, the increase in NGF, BDNF and NT-3 protein expression and NGF secretion following OGD also suggest LC cells and ONH astrocytes may be a paracrine source of neurotrophins for RGCs. | Background Glaucoma is an optic neuropathy defined by characteristic optic nerve head and associated visual field changes. Nearly 67 million people worldwide are believed to have glaucoma, including an estimated 2.2 million in the USA [ 1 , 2 ]. Primary open-angle glaucoma (POAG) is the most common form of glaucoma accounting for virtually half of all cases [ 3 ]. The visual field changes associated with POAG are due to the loss of retinal ganglion cells (RGCs), which is proposed to occur via apoptosis [ 4 , 5 ]. There is evidence that ischemia contributes to RGC loss in glaucoma. Abnormal optic nerve head (ONH) and retinal blood flow has been observed in glaucoma, and retinal ischemia results in RGC loss [ 6 - 11 ]. In addition, excitotoxicity due to elevated glutamate levels, which occurs following ischemia, can cause RGC death [ 12 - 16 ]. However, not all cellular responses to ischemia are deleterious. The expression of "protective factors", including neurotrophins (NTs), by neurons and glia within the CNS has been shown to increase following ischemia [ 17 - 19 ]. Neurotrophins are polypeptide growth factors involved in the development and maintenance of neurons, as well as non-neuronal cells. Included in this family of trophic factors are nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4 (NT-4). Neurotrophin signaling occurs via two types of receptors including (a) tyrosine kinase high affinity Trk receptors and (b) the low affinity p75 receptor [ 20 ]. The Trk receptors include Trk A, Trk B and Trk C that bind NGF, BDNF and NT-4, and NT-3 respectively [ 21 ]. Truncated isoforms of Trk B and Trk C that lack the tyrosine kinase domain have been identified and their function at this time is unknown, although there is evidence that these receptors interfere with full-length trk signaling through ligand sequestration [ 22 - 24 ]. In addition to their localization at axon terminals, Trk receptors have been localized at neuronal cell bodies, at dendritic projections, and along axons [ 25 - 27 ]. Discrete signaling pathways can be activated and distinct biological responses elicited in neurons depending on the location of Trk receptor stimulation indicating neurons respond not only to retrograde NT sources, but also to paracrine and autocrine sources [ 28 - 31 ]. The lamina cribrosa (LC) region of the optic nerve head (ONH) is composed of connective tissue plates that align to form a sieve-like structure that guides and protects RGC axons as they exit the eye to form the optic nerve. Two major cell types have been isolated from the human LC and include ONH astrocytes and LC cells [ 32 - 35 ]. We have previously demonstrated the expression of NTs and their Trk receptors by cells isolated from the human LC [ 36 ]. We also reported that mRNA and protein for the low affinity p75 receptor are not expressed by cells isolated from the human ONH. In addition we have shown that LC cells and ONH astrocytes can respond to exogenous NTs via Trk phosphorylation resulting in cell proliferation and secretion of NTs [ 37 ]. Due to the proximity of LC cells and ONH astrocytes to RGC axons within the LC, these cells could serve as a paracrine source of NTs for RGCs. The administration of exogenous NTs has been shown to protect neurons from ischemic damage [ 38 , 39 ] suggesting endogenous NT sources could also be neuroprotective. A recent report by Rudzinski et al. demonstrated that ocular hypertension increased NGF and BDNF expression within the retina [ 40 ]. Furthermore, Trk A expression in the retina was increased following elevated intraocular pressure (IOP), and this increase was observed in RGCs. Cui et al. observed an increase in Trk A, Trk B and Trk C expression in RGCs following optic nerve injury [ 41 ], again suggesting a neuroprotective role for NTs in RGC injury. In POAG, the laminar plates of the LC are compressed and bow backward from the sclera producing an excavated and exaggerated optic cup [ 42 ]. Because capillaries within the LC are located in the connective tissue plates [ 43 ], elevated IOP would likely compromise the blood flow to the LC. Ischemic insults during the progression of POAG could increase the expression and/or secretion of NTs from LC cells and ONH astrocytes, thereby increasing paracrine and/or autocrine NT signaling within the LC. Given that ischemia can be a component of ocular hypertension, we used oxygen-glucose deprivation (OGD) as an acute model of in vitro ischemia and examined the expression of NTs and their receptors by cultured LC cells and ONH astrocytes following OGD. We are aware that the model used (oxygen and glucose deprivation) is not a perfect model for what occurs in the glaucomatous optic nerve head. However, this acute model was an attempt to mimic the end results of a chronic condition, and as a "first step" we felt this model was adequate for examining the response to injury in these cell types. Results Cell number following oxygen-glucose deprivation The LC and ONH astrocyte cell lines used in this study have been previously characterized and described [ 36 ]. Preliminary studies examining cell survival following anoxia, hypoxia, hypoglycemia or serum withdrawl demonstrated LC cells and ONH astrocytes were resistant to hypoxia and serum withdrawl alone (data not shown). To approach what is occurring in vivo, we used the more acute oxygen-glucose deprivation (OGD) model to examine NT and trk expression, and NT signaling in cells from the ONH. To determine an exposure of OGD that would result in cellular changes while allowing a majority of cells to remain viable, we examined cell number at various time points of OGD using an assay that estimates cell number based on cell metabolism. The oxygen level within the anoxic incubator was determined to be below detectable levels as measured using an Oxygen Test Kit (Bacharach Inc., Pittsburgh, PA). Lamina cribrosa and ONH astrocyte cell metabolism/cell number following OGD is shown in Figure 1 . Lamina cribrosa cell metabolism/cell number did not decrease significantly until 72 hours OGD exposure. At this time point a 40% decrease in LC cell metabolism/cell number was observed. A 20% decrease in LC cell metabolism/cell number was observed following 24 or 48 hours recovery when compared to the controls. ONH astrocyte cell metabolism/cell number also decreased 20–30% following 24–48 hours OGD. A statistically significant 50% decrease in cell metabolism/cell number was observed after 72 hours of OGD. Recovery increased ONH astrocyte cell metabolism/cell number slightly when compared to OGD. We chose to collect protein and conditioned media following (a) 48 hours exposure to in vitro ischemia and (b) 48 hours in vitro ischemia plus 24 hours recovery. We chose these time points since greater than 80% of LC cells and ONH astrocytes were present indicating that a majority of cells were viable. Neurotrophin protein expression following oxygen-glucose deprivation The expression of NT protein in LC cells and ONH astrocytes following OGD is shown in Figures 2 and 3 . Multiple isoforms for all four NTs were observed in LC cells and ONH astrocytes. These isoforms most likely represent proneurotrophins, premature forms of the NTs that have recently been shown to possess biological activity [ 44 ]. A representative blot for each NT is shown. Band density reported as mean percent of the control ± SEM of three cell lines is shown graphically beneath the blots. Western blots for NTs and trks were stripped and re-probed for β-actin to ensure equal loading. Expression of β-actin in LC cells and ONH astrocytes was not significantly influenced by OGD or recovery from OGD. The overall average β-actin band density reported as a percent of the control ± SEM for LC cells was 108% ± 7 following OGD exposure and 96% ± 3 following recovery from OGD. Similar changes were observed in ONH astrocytes (109% ± 8 following OGD and 107% ± 7 following recovery from OGD). As seen in Figure 2 , the overall trend following 48 hours of OGD was a decrease in NT protein expression by LC cells. The exceptions to this trend were NGF (58 kDa), BDNF (78 kDa), and NT-3 (57 kDa), of which only NT-3 (57 kDa) demonstrated a statistically significant increase in protein expression following OGD. Of the remaining NT isoforms, BDNF (67 kDa) and NT-4 (69 kDa) demonstrated a statistically significant decrease in expression following OGD. A recovery period of 24 hours in growth media and an aerobic environment following 48 hours OGD resulted in elevated NT protein expression by LC cells, however only BDNF (67 kDa) expression levels were statistically significant. Following 48 hours of OGD, ONH astrocytes (Figure 3 ) demonstrated increased protein expression of NGF (58 kDa), NT-3 (57 kDa) and BDNF (78 kDa); only BDNF (78 kDa) was statistically significant. In addition, the expression of NGF (71 kDa) and BDNF (67 kDa) was decreased to a statistically significant level following OGD. Increased NT protein expression was the trend in ONH astrocytes allowed to recover from OGD for 24 hours in growth media and an aerobic environment. The exceptions were BDNF (78 kDa) and NT-4 (54 kDa), which were slightly lower than control levels. Of the NT isoforms where protein expression increased during recovery, only NGF (71 kDa) demonstrated a statistically significant increase. Western blot densitometry results for LC cells and ONH astrocytes are summarized in Table 1 . Trk receptor protein expression following oxygen-glucose deprivation Figure 4 represents the expression of Trk receptor protein in LC cells following OGD or recovery from OGD. Data are presented as described in the previous section. Protein expression of Trk B and Trk C receptors decreased to a statistically significant level in LC cells following 48 hours of OGD. In contrast, a statistically significant increase in the expression of Trk A protein was observed following exposure to OGD. Recovery from OGD resulted in a statistically significant decrease in Trk A and Trk C expression, while Trk B and truncated Trk B protein expression was elevated, but not to a statistically significant level. The expression of Trk receptor protein by ONH astrocytes following OGD and recovery from OGD is shown in Figure 5 . A statistically significant decrease in the expression of Trk A, Trk C and truncated Trk B protein by ONH astrocytes was observed following OGD. Trk B protein expression was also decreased, but not to a significant level. Recovery from OGD increased Trk C and truncated Trk B receptor protein expression in ONH astrocytes compared to OGD alone. However, Trk C expression was still decreased to a statistically significant level. Trk A and Trk B protein expression was increased in ONH astrocytes allowed to recover from OGD, although only Trk A expression was statistically significant. Phosphorylated Trk receptor protein expression following oxygen-glucose deprivation The expression of phosphorylated Trk receptor protein following OGD and recovery from OGD is shown in Figure 6 . Data are presented as described above. Trk phosphorylation indicates the activation of Trk receptors following NT binding. The antibody used was a phospho-pan Trk antibody that recognizes phosphorylated forms of Trk A, Trk B and Trk C receptors. Four phospho-Trk isoforms (148 kDa, 120 kDa, 80 kDa, and 72 kDa) were detected in LC cell and ONH astrocyte controls. The 120 kDa and 72 kDa phospho-Trk isoforms most likely represent phosphorylated Trk A and Trk B respectively [ 37 ]. The remaining phospho-Trk isoforms could represent phosphorylated forms of Trk A, B or C. Overall, exposure to OGD resulted in decreased phospho-Trk protein expression by LC cells and ONH astrocytes. Expression of the 148 kDa phospho-trk isoform was decreased to a statistically significant level in both LC cells and ONH astrocytes, as was the 120 kDa and 80 kDa isoforms in ONH astrocytes. The only increase in phospho-Trk receptor protein expression following OGD was observed in LC cells. Protein expression of the 120 kDa phospho-Trk receptor isoform was increased 60% over the controls in LC cells, which was statistically significant. Recovery from OGD resulted in increased protein expression of phospho-Trk receptor isoforms in LC cells and ONH astrocytes when compared to OGD alone, with the exception of the 120 kDa isoform in LC cells, which decreased toward control levels. Although its expression increased compared to OGD alone, expression of the 148 kDa isoform was still decreased to a significant level. Interestingly, the 120 kDa phospho-trk isoform was again the only isoform whose expression was increased above control levels. This 100% increase was observed in ONH astrocytes following recovery from OGD and was statistically significant. Neurotrophin secretion following oxygen-glucose deprivation The secretion of NGF and BDNF following OGD and recovery from OGD is shown in Figure 7 . Data are presented as mean percent of the control ± SEM of three LC and three ONH astrocyte cell lines. The secretion of NGF by LC cells and ONH astrocytes was increased over 200% and 150% following OGD, respectively. This increase in NGF secretion by cultured cells of the human LC was statistically significant. In contrast, BDNF secretion by both cell types was decreased 60 to 90% following OGD, which was statistically significant. Although recovery from OGD returned NGF and BDNF secretion by LC cells toward controls levels, a statistically significant decrease in BDNF secretion by ONH astrocytes was observed. The secretion of NT-3 or NT-4 was not detected by either cell type under any condition. Discussion In this study we examined the protein expression of NTs and Trk receptors by LC cells and ONH astrocytes following OGD, an in vitro model of ischemia. Lamina cribrosa cell and ONH astrocyte responses to OGD and recovery from OGD are summarized in Tables 2 and 3 . Lamina cribrosa cells and ONH astrocytes increased NGF, BDNF and NT-3 protein expression following OGD. NGF secretion by these cells was also increased by OGD at the time points tested. Exposure to OGD decreased Trk protein expression and activation in LC cells and ONH astrocytes, with the exception of Trk A in LC cells. Recovery from OGD resulted in most NT and Trk receptor protein expression returning toward control levels in LC cells and ONH astrocytes. However, Trk A expression and activation in ONH astrocytes remained significantly elevated over control levels. Overall, this study suggests that paracrine and/or autocrine NT signaling is stimulated in cells from the ONH following an ischemic insult. Alternatively, NT release by ONH cells may act to protect RGCs from ischemic injury. Ischemia due to elevated IOP during POAG may cause changes within the ONH and contribute to RGC loss [ 6 - 11 ]. As a response to ischemia, neurons and glia have been shown to increase the expression of NTs [ 17 - 19 ]. Local NT sources for RGCs within the retina and LC could potentially protect these neurons during periods of ONH ischemia in POAG. The recent report of Rudzinski et al. [ 40 ] is of interest as they demonstrated an up-regulation of both NGF and Trk A after 7 days of ocular hypertension, and a sustained up-regulation of BDNF after 28 days elevated IOP. We have previously demonstrated NT expression and secretion by LC cells and ONH astrocytes [ 36 ]. The LC region of the ONH and the cells that reside there may be subject to ischemic injury during the progression of POAG. Therefore, we examined cell number as determined by cell metabolism following OGD to determine if LC cells and ONH astrocytes could survive ischemic insult. Lamina cribrosa cell metabolism/cell number remained within 15% of the controls until 72 hours of OGD, whereas ONH astrocyte cell metabolism/cell number decreased 20–30% over the same exposure time. Lamina cribrosa and ONH astrocyte cell metabolism/cell number returned to within 20% of the controls following recovery from OGD. These results suggest LC cells and ONH astrocytes can survive ischemic injury and therefore could be a potential source of neuroprotective factors. Neurons, including RGCs, express Trk receptors not only at the axon terminal and cell body, but also along their axons [ 25 - 27 ] suggesting RGCs could bind NTs provided by cells of the ONH. We examined the expression and secretion of NTs by LC cells and ONH astrocytes following OGD to determine if these cells could potentially protect RGCs from ischemic injury. The expression of NGF, BDNF and NT-3 by LC cells and ONH astrocytes increased after exposure to OGD. Examination of NT mRNA levels in LC cells and ONH astrocytes following OGD would determine whether this is due to an up-regulation in expression or to increased processing of proNTs. Interestingly, only NGF secretion by LC cells and ONH astrocytes was increased by OGD. It is possible that BDNF and NT-3 are secreted by LC cells and ONH astrocytes following OGD, but at time points other than those examined or at lower detection levels. Overall, the responses to OGD by LC cells and ONH astrocytes with respect to the expression of NTs appeared favorable to RGCs. Neurotrophin expression was increased in both cell types, as was NGF secretion. Thus it appears LC cells and ONH astrocytes could provide RGCs with neuroprotective factors following ischemic injury. The expression of Trk receptors (both full length and truncated) by LC cells, ONH astrocytes or other cells within the LC could limit NT availability to RGCs [ 22 - 24 ]. Therefore, we examined Trk protein expression and activation in LC cells and ONH astrocytes following OGD. Trk receptor expression was decreased in LC cells and ONH astrocytes following OGD, with the significant exception of Trk A in LC cells. As an indication of NT signaling, we examined the expression of phosphorylated Trk receptors in LC cells and ONH astrocytes following OGD. Phospho-Trk A expression in LC cells was increased after OGD, while all other phospho-Trk expression was decreased. The up-regulation of autocrine NGF signaling in LC cells (e.g. NGF secretion and Trk A expression) could explain the differences observed between LC and ONH astrocyte cell number in following OGD. In a previous report we demonstrated that exogenous NGF increased LC cell number [ 37 ]. As LC cells do not express p75, this response is most likely due to Trk A activation. Together these data suggest Trk expression by ONH astrocytes would not interfere with NT availability to RGCs during ischemia. In contrast, increased expression of Trk A in LC cells resulted in increased Trk A activation, implying NGF expression in these cells during ischemia may be self protective rather than protective toward RGCs. There is evidence that reperfusion following ischemia is actually more detrimental to cells than ischemia itself [ 45 , 46 ]. To determine if cells from the LC could provide RGCs with NT support during reperfusion of the ONH in POAG, we examined NT and Trk receptor expression following recovery from OGD. ONH astrocytes could be a source of NTs for RGCs during reperfusion of the ONH as an increase in NGF protein expression was observed in these cells after recovery from OGD. However, recovery from OGD also increased Trk A expression and activation in ONH astrocytes, implying these cells up-regulate autocrine/paracrine NGF signaling during recovery from an ischemic event. In addition, Trk B receptor expression by LC cells and ONH astrocytes increased after recovery from OGD suggesting NT availability to RGC axons within the LC may be compromised during ONH reperfusion. Based on these results, LC cells and ONH astrocytes would be unable to provide RGCs with NTs during reperfusion of the ONH. Increased NT expression by LC cells and ONH astrocytes during ONH ischemia could protect RGCs from injury; however, as blood flow was restored NT expression by LC cells and ONH astrocytes would decrease to normal levels, leaving RGCs vulnerable to injury. Neurotrophin expression by LC cells and ONH astrocytes may be beneficial to RGCs during ischemia, but may be unable to promote the survival of these neurons during reperfusion. In conclusion, we have demonstrated that LC cells and ONH astrocytes increase the expression of NGF, BDNF and NT-3 protein following OGD, which may be neuroprotective for RGCs. Neurotrophins expressed by LC cells and ONH astrocytes following OGD or recovery from OGD bind and activate Trk receptors expressed by these cells. Increased NT signaling within LC cells and ONH astrocytes could increase cell survival following ischemic injury, but may compromise RGC survival during reperfusion. Further studies examining the expression of NTs and Trk receptors following hypoxia or transient ischemic insults would provide a better model for ischemic injury in POAG. Using this model, RGCs could be co-cultured with LC cells or ONH astrocytes to determine the neuroprotective effects of NTs during ischemic injury. By better understanding NT signaling within the LC under normal and injurious conditions, new strategies involving these factors could be developed to better treat patients with POAG. Conclusions Lamina cribrosa cells and ONH astrocytes respond to conditions that mimic ONH ischemia by increasing NGF, BDNF and NT-3 protein expression and NGF secretion. Increased protein expression of Trk receptors and phosphorylated Trk receptors by LC cells and ONH astrocytes following OGD and recovery from OGD respectively suggest paracrine and/or autocrine NT signaling occurs within the ONH following ischemic injury. Lamina cribrosa cells and ONH astrocytes may be a paracrine source of NGF, BDNF and/or NT-3 for RGCs, especially during ischemic injury within the ONH throughout POAG progression. Methods Materials DMEM and fetal bovine serum (FBS) were purchased from HyClone Labs, Logan, UT. The following materials were purchased from Gibco BRL Life Technologies, Grand Island, NY; glucose free DMEM, L-glutamine, penicillin/streptomycin and fungizone (amphotericin B). Costar 96-well plates and Nunc ELISA/EIA 96 well Maxisorp plates were purchased from Fisher Scientific, Pittsburgh, PA. Polyclonal antibodies to Trk A, Trk B, Trk C, truncated Trk B (Trk B.T) and phosphorylated Trk were purchased from Santa Cruz Biotechnology Inc, Santa Cruz, CA. CellTiter 96 ® Aqueous Non-Radioactive Cell Proliferation Assays and Emax™ ImmunoAssay Systems specific for NGF, BDNF, NT-3 and NT-4 were purchased from Promega Corporation, Madison, WI. Lamina cribrosa and ONH astrocyte cell culture Lamina cribrosa and ONH astrocyte cell lines were obtained from human LC explants from separate donors as described previously [ 36 ]. Cells were cultured in Ham's F-10 Media (LC cells, JRH Biosciences, Lenexa, KS) or DMEM (ONH astrocytes) supplemented with 10% FBS, L-glutamine (0.292 mg/ml), penicillin (100 units/ml)/streptomycin (0.1 mg/ml), and amphotericin B (4 μg/ml) as previously described. Cells were passaged using a 0.25% trypsin solution (Sigma-Aldrich, St. Louis, MO). All cultures were maintained in 5% CO 2 /95% air at 37°C and media was changed every 2 to 3 days. Characterization of these cells was performed as described previously [ 36 ]. Cells expressing α-smooth muscle actin that did not express glial fibrillary acidic protein (GFAP) were characterized as LC cells [ 32 , 33 , 36 ]. Cells expressing GFAP and neural cell adhesion molecule (N-CAM) were characterized as ONH astrocytes [ 34 - 36 ]. Both cell types expressed extracellular matrix proteins, such as collagen I, collagen III, collagen IV and elastin [ 32 , 33 , 36 ]. Adult cell lines from donors whose ages ranged from 39 years to 90 years were used in the following experiments. Oxygen-glucose deprivation Preliminary studies examining cell survival following anoxia, hypoxia, hypoglycemia or serum withdrawl demonstrated LC cells and ONH astrocytes were resistant to hypoxia and serum withdrawl alone (data not shown). To approach what is occurring in vivo, we used the more acute oxygen-glucose deprivation (OGD) model to examine NT and trk expression and NT signaling in cells from the ONH. Preconfluent, age-matched adult LC cells and ONH astrocytes were treated with serum free media for 24 hours. Oxygen-glucose deprivation (OGD) was achieved by culturing LC cells and ONH astrocytes in glucose free serum free DMEM in an anoxic incubator (95% N 2 /5% CO 2 ) for 48 hours. The oxygen level within the anoxic incubator was measured using a Oxygen Test Kit (Bacharach Inc., Pittsburgh, PA) and was determined to be below detectable levels. Recovery following OGD was achieved by placing cells in OGD conditions for 48 hours and then allowing them to recover in growth media (Ham's F-10 Media or DMEM plus 10% FBS) and an aerobic environment (95% air/5% CO 2 ) for 24 hours. Cells cultured in growth media and an aerobic environment for served as controls. Determination of cell number based upon cell metabolism following oxygen-glucose deprivation Adult LC cells and ONH astrocytes were trypsinized, counted using a hemacytometer and plated into Costar 96-well plates at a density of 1,000 cells/well. Cells were allowed to attach overnight and were then placed in serum free media for 24 hours. Lamina cribrosa cells and ONH astrocyte were exposed to OGD as described above for 24, 48 or 72 hours. Recovery following OGD was achieved by placing cells in OGD conditions for 24 or 48 hours and then allowing them to recover in growth media and an aerobic environment (95% air/5% CO 2 ) for 24 hours. Cells cultured in growth media and an aerobic environment for served as controls. Cell number was estimated using the CellTiter 96 ® Aq ueous Non-Radioactive Cell Proliferation Assay which measures product from a metabolic process to estimate cell number. Following exposure to OGD or recovery from OGD, media was removed and replaced with 100 μl of serum free media. Twenty microliters of MTS/PMS solution was added to each well. Plates were incubated at 95% air/5% CO 2 at 37°C for 1 hour, at which time the absorbance at 490 nm was read using a SpectraMax ® 190 microplate reader and Softmax ® Pro (Molecular Devices Corporation, Sunnyvale, CA). Metabolically active cells convert MTS into formazan, which is soluble in aqueous solutions. The quantity of the formazan product measured by the amount of absorbance at 490 nm is therefore directly proportional to the number of living cells. Cell metabolism/cell number per well was calculated from a standard curve generated using known amounts of cells per well. A standard curve was generated for each cell line assayed. Three LC cell lines and three ONH astrocyte cell lines were assayed. The entire experiment, including standard curves, was repeated twice. Changes in cell metabolism/cell number following OGD and recovery from OGD were reported as a percent of the control ± SEM. Protein extraction and western blot analysis Cellular protein was collected in lysis buffer modified from Watson et al. [ 47 ] [20 mM Tris (pH 7.4), 137 mM NaCl, 1% NP40, 10% glycerol, 48 mM sodium fluoride, 16 mM sodium pyrophosphate, 1 mM PMSF, 20 μM leupeptin, 10 μg/ml aprotinin, and 1 mM sodium orthovanadate (10 μl/ml)]. Protein concentration was measured using the Bio-Rad D c Protein Assay System (Bio-Rad Laboratories, Richmond, CA). Cellular lysate (50 μg) was separated on denaturing polyacrylamide gels and then transferred by electrophoresis to nitrocellulose membranes. Blots were processed using primary antibodies and the Western Breeze Chemiluminescent Immunodetection System (Invitrogen, Carlsbad, CA). Blots were then exposed to Hyperfilm-ECL (Amersham, Arlington Heights, IL) for various times depending on the amount of target protein present. The density (O.D. × mm 2 ) of unsaturated bands was measured using the Discovery Series scanner and the Diversity One program from pdi (Huntington, NY) and a digital Venturis FP466 computer (Compaq, Houston, TX). Western blots for NTs and trks were stripped and re-probed for β-actin to ensure equal loading. Conditioned media and immunoassays Conditioned media was collected and concentrated using Millipore Centriplus YM-3 Centrifugal Filter Devices (Millipore Corporation, Bedford, MA). Emax™ ImmunoAssay Systems specific for NGF, BDNF, NT-3 and NT-4 (Promega) were performed according to manufacturer's instructions. Conditioned media was added to Nunc ELISA/EIA 96 well Maxisorp plates coated with anti-NT polyclonal antibodies. Secreted NT was detected by treating the plates with the respective NT monoclonal antibody followed by a horseradish peroxidase conjugated secondary antibody. Enzyme substrate was added to generate a color product whose absorbance was read at 450 nm. A NT standard included in each assay was used to generate a standard curve that was used to calculate the amount of secreted NT per well. The amount of secreted NT per sample was normalized to total protein per sample. Samples were assayed in triplicate. Conditioned media from three LC cell lines and three ONH astrocyte cell lines were assayed. Each immunoassay was repeated twice. Changes in NT secretion following OGD and recovery from OGD were reported as a percent of the control ± SEM. Statistical analysis Cell metabolism/cell number, western blot densitometry and immunoassay data were analyzed using one way analysis of variance (ANOVA) followed by validation using Student-Newman-Keuls tests. The MedCalc ® statistical package, version 7.4.41 [ 48 ] was used for statistical analysis. Significance values were adjusted in accordance with Bonferroni's correction for multiple tests [ 49 ]. Authors' contributions WL carried out tissue culture, cell proliferation assays, Western blotting, and immunoassays. WL, AC, and RW participated in design of the study, interpretation of the results, and in the writing and revision of the manuscript. All authors read and approved the final manuscript. This study is taken in part from a dissertation submitted to the UNT Health Science Center in partial fulfillment of the requirements for the degree Doctor of Philosophy for WL. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC539236.xml |
544587 | Epitope definition by proteomic similarity analysis: identification of the linear determinant of the anti-Dsg3 MAb 5H10 | Background Walking along disease-associated protein sequences in the search for specific segments able to induce cellular immune response may direct clinical research towards effective peptide-based vaccines. To this aim, we are studying the targets of the immune response in autoimmune diseases by applying the principle of non-self-discrimination as a driving concept in the identification of the autoimmunogenic peptide sequences. Methods Computer-assisted proteomic analysis of the autoantigen protein sequence and dot-blot/NMR immunoassays are applied to the prediction and subsequent validation of the epitopic sequences. Results Using the experimental model Pemphigus vulgaris /desmoglein 3, we have identified the antigenic linear determinant recognized by MAb 5H10, a monoclonal antibody raised against the extracellular domain of human desmoglein-3. The computer-assisted search for the Dsg3 epitope was conducted by analyzing the similarity level to the mouse proteome of the human desmoglein protein sequence. Dot-blot immunoassay analyses mapped the epitope within the sequence Dsg3 49–60 REWVKFAKPCRE, which shows low similarity to the mouse proteome. NMR spectroscopy analyses confirmed the specificity of MAb 5H10 for the predicted epitope. Conclusions This report promotes the concept that low level of sequence similarity to the host's proteome may modulate peptide epitopicity. | Introduction In the last decades, several computer-driven algorithms have been devised to take advantage of the linear representation of protein sequence information to search for epitopic motifs [ 1 - 5 ]. These algorithms search the amino acid sequence of a given protein for characteristics believed to be common to antigenic peptides, locating regions that are likely to induce cellular immune response. Given the rapid expansion of proteomic sequence data, the application of these algorithms to disease-associated proteins may direct research to specific segments of disease-associated proteins and thus potentially reduce the time and effort needed to develop effective vaccines [ 6 , 7 ]. We are using in silico technology platforms to identify epitopic peptide sequence(s) from disease-associated-antigens by following the hypothesis that peptide epitopicity might be regulated by the peptide similarity level to the host's proteome, in addition to other factors such as MHC binding potential [ 8 - 10 ]. As a scientific rationale, we follow the criterion that immune system might be allowed/forced to respond only to rarely encountered/never seen antigenic sequences. Accordingly, we explain the non-immunogenicity of tumor-associated-antigens as due to high level of similarity of oncoprotein sequences to self-proteome [ 8 , 9 , 12 ]. In this context we have tested here the possibility that endogenous, normal, housekeeping self-proteins harbouring sequences with little or no similarity to the collective host proteome might be epitopic targets in autoimmune responses. Self-reactivity and autoimmunity are processes related to the breakage of self-tolerance that can be distinguished by their different clinical outcome. The transition from self-reactivity to the autoimmune pathology appears to be mediated by a complex network of overlapping phenomena that comprehend epitope spreading, uncovering of cryptic epitopes, natural autoantibodies production, cross-reactivity, microchimerism, altered B lymphocyte function, inflammation, etc. [ 13 ]. In our labs, we are studying the autoantibody profile in Pemphigus vulgaris (PV). PV is an autoimmune bullous skin disease characterized by autoantibodies to a desmosomal adhesion molecule, the cadherin desmoglein-3 (Dsg3) [ 14 ]. Dsg3 represents an optimal autoantigen for studying the relationship between similarity level and immune responses. Indeed the PVA Dsg3 is a highly conserved protein, and the human and mouse forms present 71.6% of identity. Therefore, this protein allows to analyze the sequence specificity of the reaction PVA-autoantibody by using murine monoclonal antibodies. Moreover, our interest to study the autoantibody response in this autoimmune disease also stems from the following considerations: i) so far, notwithstanding the Dsg3 linear structure, attention has been focused mainly on conformational Dsg3 epitopes [ 15 - 18 ] and there is a lack of data on the occurrence and fine molecular characterization of linear Dsg3 epitopes; ii) although the precise pathological implications of anti-Dsg autoantibodies are not fully elucidated [ 19 - 23 ], it seems likely that a spectrum of autoantibodies, differing in number and type, might contribute to the autoimmune pathology in PV [ 24 ]. Given these premises, the development of new experimental approaches that might lead to the exact definition of linear PVA epitopes is desirable. As a first attempt to a computer-driven individuation of defined linear epitopic sequences of Dsg3, we describe here the identification of the linear epitope of the MAb 5H10 raised in BALB/c mice against the extracellular domain (EC1/EC2, aa1-212) of human Dsg3. Materials and methods Computer-assisted analyses The amino acid sequence corresponding to accession number P32926, SWISS-PROT, was used in the similarity analysis of the extracellular domain of human Dsg3 (EC1 to part of EC2, amino acid 1–212) to the mouse proteome. The Dsg3 sequence under analysis was dissected into pentamer motifs, that were probed for sequence similarity to mouse proteome by using PIR non-redundant reference protein database and peptide match program [ 25 ]. The search was conducted against mouse complete genome for a total of 78435 sequences, and includes hypothetical/unidentified proteins sequences. Antibodies Anti-Dsg3 MAb 5H10 and 5G11, raised in BALB/c mice, were a generous gift of Dr. Margaret Wheelock, University of Toledo, Ohio. Both MAbs recognize a linear determinant and have been described in detail [ 26 ]. Briefly, MAb 5H10 has been shown to recognize the amino terminal part of the extracellular domain of Dsg3 (EC1 to part of EC2, amino acid 1–212). MAb 5G11 reacts with the carboxyl terminus of the EC domain of Dsg3 (part of EC4 to EC5, amino acid 446–613). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG-specific Abs was from Sigma, Chemical Co., St.Louis, Mo. Synthetic peptides Peptides were synthesized using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase peptide synthesis. Peptide purity (>90%) was controlled by analytical HPLC, and the molecular mass of purified peptides confirmed by fast atomic bombardment mass spectrometry. The EC1/EC2 Dsg3 36–45 EEMTMQQAKR, Dsg3 49–60 REWVKFAKPCRE, and Dsg3 173–187 NSLVMILNATDADEP peptides were obtained from PeptidoGenic Research & Co., Livermore, CA., and used in dot-blot immunoassay experiments. Human recombinant Dsg3 protein was used as further control [ 27 ]. The 15 N-labelled synthetic peptide Dsg3 49–60 R EW V K FA KPC R E (where 15 N-labelled amino acid residues are underlined) was from Primm srl, Milan, Italy. Peptides were dissolved in 0.9% NaCl, aliquoted and stored at -20°C. Dot immunoassay Nitrocellulose membrane (0.2 μm pore size, BioRad Laboratories, Hercules, CA) was pretreated for 10 min with 1.0% glutaraldehyde. Synthetic peptides were spotted on the activated membranes, left to dry at RT and exposed to UV rays for 10 min. Membranes were incubated for 1 h in phosphate-buffered saline/0.05% (v/v) Tween 20 (PBST) containing 4% bovine serum albumin (BSA), and then with the primary MAb (1:800) for further 2 h. Membranes were washed for 10 min with PBST (x3) and incubated in PBST/4% BSA with HRP-conjugated affinity-purified goat anti-mouse IgG (1:1000) for 1 h. Membranes were washed in PBST for 5 min (x3), in PBS for 5 min (x3) and immunoblots were developed using the enhanced chemiluminescence detection assay (Renaissance, NEN™ Life Science Products, Boston, MA.) following supplier's instructions. NMR spectroscopy NMR spectra of the reaction between the synthetic 15 N-labelled peptide Dsg3 49–60 R EW V K FA KPC R E (where 15 N-labelled amino acid residues are underlined) and MAb 5H10 (raised against EC1/EC2 from Dsg3) or 5G11 (raised against EC4/EC5 from Dsg3) were recorded at 298°K on a Bruker Avance DRX500WB spectrometer. The spectra were acquired by heteronuclear single quantum correlation (HSQC) experiments that correlate the chemical shift of proton with the chemical shift of the directly bonded nitrogen. Specifically, the two-dimensional 1 H- 15 N inverse detected correlation spectra were acquired by the gradient pulse sensitivity improved Bruker automation program INVIETGPSI [ 28 ]. The 1 H acquisition dimension was collected with a spectral width of 5 ppm, centered at 7.6 ppm, using 4096 datapoints for each of the total 32 scans/expt. Spectral width in the indirect detected 15 N dimension was 200 ppm, centered at 90 ppm, obtained with a total of 1024 points. The spectra were processed by XwinNMR program, version 2.6, running on an INDY R5000 Silicon Graphics Workstation. Chemical shift values were referenced to sodium tetrasilyl propionate (TSP) ( 1 H; 0.000 ppm) and external neat nitromethane ( 15 N; 380.23 ppm) standards. To avoid interferences of TSP standard with peptide samples, the instrument scale was preliminarily calibrated in parallel experiments with samples containing the peptide to be analysed plus 0.2 mg TSP. We used chemical shift statistics from the full BioMagResBank database, where the calculated statistics are derived from a total of 559392 chemical shifts (website: ). Sequence-specific correction factor tabulations were applied to backbone 1 H and 15 N resonances [ 29 ]. Two-dimensional correlated spectroscopy spectra of MAb-peptide complex were obtained using a 1:2 stoicheiometric molar ratio (peptide:MAb, 0.1:20, mg/mg). That is, NMR samples contained either 0.1 mg free R EW V K FA KPC R E peptide, or 20 mg MAb 5H10 complexed with 0.1 mg R EW V K FA KPC R E peptide, or 20 mg MAb 5G11 complexed with 0.1 mg R EW V K FA KPC R E peptide, in 0.5 ml aqueous solution H 2 O/D 2 O (9:1, v/v). Results Selection of Dsg3 peptide sequences with low similarity to the mouse proteome The EC1/EC2 domain (aa1-212) of human Dsg3 was dissected into 5-mer sequences and analyzed for similarity to mouse proteins. The pentamers used for epitope scanning were offset by one residue, i.e. overlapped by 4 residues: MMGLF, MGLFP, GLFPR, LFPRT, etc. The 5-mer sequences were probed in computer-assisted similarity analyses against the complete mouse genome sequences. Fig. 1 reports the profile we obtained by representing the number of matches to mouse proteome over the sequential pentamer peptides. It can be seen that the EC1/EC2 Dsg3 protein sequence presents stretches scarcely represented in the mouse proteome. Among the low-similarity EC1/EC2 Dsg3 peptide fragments, the Dsg3 49–60 REWVKFAKPCRE sequence was the longest (12 amino acid residues long) with the lowest number of matches to the murine proteome (number of matches to murine proteome = 9). The Dsg3 49–60 REWVKFAKPCRE sequence and a second low-similarity fragment, Dsg3 36–45 EEMTMQQAKR (number of matches to murine proteome = 12), were selected to test our hypothesis. Figure 1 Molecular mimicry between the EC1/EC2 of human Dsg3 and mouse proteome . The EC1/EC2 Dsg3 sequence (aa1-212) was scanned for matches to mouse protein sequences by using pentamers offset by one residue. The arrow indicates the longest stretch having the lowest number of matches. Dot-blot immunoassay The two low similarity peptides selected as described above were synthetised in order to be assayed in immunodot-blot analyses to identify the linear determinant of the MAb 5H10. In addition, the synthetic Dsg3 173–187 NSLVMILNATDADEP peptide (matches to murine proteome = 109) was available and served as a high similarity peptide control. The three synthetic peptides are described in Fig. 2 . The MAb 5G11 with specificity to the terminal EC portion of Dsg3, amino acid 446–613 [ 26 ] was used as a primary antibody control. The immunodot-blot experimental result is illustrated in Fig. 3 . It can be seen that MAb 5H10 recognized peptide n. 2, i.e. the Dsg3 49–60 REWVKFAKPCRE sequence having a lowest level of similarity to the mouse proteome (see Fig. 1 ). No reaction was monitored using MAb 5G11. Figure 2 Similarity scanning on the human Dsg3 peptide sequences selected for immunoassay analysis with murine anti-EC1/EC2 Mab 5H10 . Matching analysis to the murine proteome was performed using as probes pentamers offset by one residue as described under Methods. Peptide: 1) Dsg3 36–45 EEMTMQQAKR; 2) Dsg3 49–60 REWVKFAKPCRE; 3) Dsg3 173–187 NSLVMILNATDADEP. Figure 3 Identification of the epitopic sequence recognized by mouse anti-Dsg3 1–212 MAb 5H10 . Dot-blot immunoassay was performed on nitrocellulose membrane spotted with human Dsg3 (10μg), or Dsg3 peptide (2.5μg) corresponding to the sequence: 1) Dsg3 36–45 EEMTMQQAKR; 2) Dsg3 49–60 REWVKFAKPCRE; 3) Dsg3 173–187 NSLVMILNATDADEP. NMR spectroscopic immunoanalysis As a further step, we carried out a parallel experimental confirmation because of the doubts of false negative/positive data intrinsic to immunoassay. To this aim, the binding of the predicted epitopic Dsg3 49–60 REWVKFAKPCRE peptide and the mouse anti-Dsg3 5H10 MAb was further verified by NMR spectroscopy. NMR spectroscopy can be used in 1) defining structure and conformation; 2) defining structure-activity relationships; 3) monitoring chemical reactions. Informations are mainly derived by measuring NMR chemical shifts. The chemical shift of a nucleus is the difference between the resonance frequency of the nucleus and a standard. This quantity is reported in parts per million (ppm). The NMR standards are molecules as tetrasilyl propionate, the signal of which is set at 0 ppm by having shielded protons. The NMR chemical shift allows for distinguishing magnetically inequivalent nuclei in a molecule, i.e. chemical shifts are a measure of the motional freedom. We utilized NMR spectroscopy in order to determine whether the predicted peptide specifically binds to the MAb 5H10. To this aim, a 15 N-labelled R EW V K FA KPC R E peptide(where 15 N-labelled amino acid residues are underlined) was synthesized. Theoretical chemical shift values of the 15 N-labelled residues in the Dsg3 49–60 R EW V K FA KPC R E peptide were calculated as described under Methods, and then were compared to the experimental values. Table 1 lists the theoretical and experimental chemical shift values of 15 N-labelled residues in the Dsg3 49–60 R EW V K FA KPC R E peptide, and their values following addition of MAb 5H10 or MAb 5G11. Table 1 Sequence-specific assignments in the 15 N-labelled Dsg3 49–60 R EW V K FA KPC R E peptide, and chemical shift changes on MAb 5H10 or 5G11 binding. 15 N-Amino acid Chemical Shift Values: Theoretical: Free peptide Experimental: Free peptide +MAb 5H10 +control MAb 5G11 1 H N 15 N 1 H N 15 N 1 H N 15 N 1 H N 15 N Arg-1 6.78 77.6 6.95 81.0 n.d. n.d. 7.05 80.5 Val-4 8.65 120.8 8.19 122.8 n.d. n.d. 8.18 122.5 Phe-6 8.42 121.7 7.99 119.2 n.d. n.d. 8.00 118.9 Ala-7 8.29 122.3 8.40 120.0 n.d. n.d. 8.44 120.2 Arg-11 6.78 77.6 7.25 81.5 n.d. n.d. 7.26 81.0 8.14 119.6 8.16 118.1 n.d. n.d. 8.15 117.8 The 15 N-amino acid position in the peptide is reported. Theoretical chemical shift values were derived from and corrected [29]. Experimental chemical shift values were derived from resonance spectra reported in Fig. 4. The average value of chemical shifts relative to the H and N atoms of α amino residue, and the H and N atoms of η amino residues are reported for Arg-11 (data from: ). The chemical shifts relative to the H and N atoms of α amino residue in Arg-1 are undetectable because of terminal position of the amino acid in the peptide. n.d., not detected. The resonance spectra of the reaction between MAb 5H10 and the 15 N-labelled Dsg3 49–60 R EW V K FA KPC R E peptide are illustrated in Fig. 4 . Each "spot" in the figure is an NMR signal representing the 1H- 15 N one-bond coupling of the labelled amino acid residues in the peptide. In Fig. 4A , the reported spots correspond to the Arg, Val, Phe and Ala selective 1 H- 15 N correlation signals of the free peptide in aqueous solution. As already illustrated in Table 1 , the expected signals are present and in basic agreement with theoretical data (see BioMagResBank database, ) following sequence-dependent correction of random coil NMR chemical shifts. [ 29 ]. The upper panel of Fig. 4A displays the two Arg cross-peak signal, i. e. the chemical shifts relative to the H and N atoms of amino residues. The lower panel in fig. 4A reports the cross-signal of 15 N-Arg-11 whereas the 15 N-Arg-1 was undetectable because of the terminal position in the peptide. Fig. 4B documents that the addition of MAb 5H10 caused the loss of 15 N-labelled residue spectral resonance signals thus indicating a complete loss of Dsg3 49–60 R EW V K FA KPC R E peptide mobility. In contrast to the total signal deletion provoked by MAb 5H10, the chemical shift values of the Dsg3 49–60 R EW V K FA KPC R E peptide remained unaltered following the addition of the anti-Dsg3 MAb 5G11 (Fig. 4C , upper and lower panels). Figure 4 1 H- 15 N NMR HSQC spectra and relative 1-D contour plots of the Dsg3 49–60 R EW V K FA KPC R E peptide 15 N-labelled at residues 7, 10, 12 and 14 (A), plus MAb 5H10 (B), or 511G (C) . Upper panels report portions of the HSQC spectra showing resonances from residues 1, 4, 6, 7, and 11. Lower panels report expanded region of the HSQC spectra showing resonances from the same residues 4, 6, 7, and 11. Discussion The present data appear of interest since, as a preliminary consideration, it has to be noted that historically the search for biologically relevant epitopic sequences has demanded and still demands complicated procedures and expensive technologies [ 30 ]. Here, the low-similarity principle we used has allowed the utilization of proteomic computational program for the exact epitope definition of a MAb directed towards a 212 amino acid sequence by using only 3 synthetic peptide reagents. Minimally, the screening of a library of at least 14 non-overlapping peptides, each 15 residues in length, would be necessary in the unassisted case. Moreover, the individuation of the Dsg3 49–60 REWVKFAKPCRE motif as the antigenic epitope of the Dsg3 EC1/EC2 domain, aa1-212, might contribute to the understanding of the autoimmune mechanisms in PV. This immunogenic sequence is hosted in the NH 2 terminal region of Dsg3, which has adhesive function in cadherins [ 31 ] and contains the major epitopes recognized by sera from PV patients [ 32 ]. Further, chimeric toxins containing the Dsg3 EC1/EC2 domain, aa1-212, have been demonstrated to downregulate anti-Dsg3 IgG-producing B cells in mice immunized with Dsg3 aa1-212 [ 26 ]. In a wider context, the epitope mapping approach here described might be helpful in defining peptide antigenicity in a wide spectrum of human diseases including cancer pathologies as well as a range of diverse autoimmune disorders such as insulin-dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, and Pemphigus vulgaris . The fine profiling of the disease-associated epitopic peptide repertoire is of particular importance in the definition of qualities as antigenicity and immunodominance, and is an essential preliminary step towards effective immunotherapeutical treatments in cancer and autoimmune pathologies. In synthesis, given the caveat that only linear sequences can be defined by the analysis of amino acid motif sharing by two or more proteins, the epitope prediction model we report could form the basis of a rapid, inexpensive and computationally driven system for the individuation of antigenic sequences that are the targets of autoimmune responses. Consequently, this proteomic strategy may serve as a general method suitable to define/distinguish/screen disease-(ir)relevant epitopes within potential autoantigens with clinical application to wide ranging human diseases where the precise targets of self-directed attack are unknown [ 33 ]. Abbreviations Dsg3, desmoglein 3; EC, extracellular; PVA, Pemphigus vulgaris autoantigen; PIR, Protein Information Resource; HSQC, heteronuclear single quantum correlation. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544587.xml |
535424 | Sleep, Appetite, and Obesity—What Is the Link? | There is a well known relationship between short sleep duration and high body mass index. A new study suggests that the missing link could be the appetite regulating hormones leptin and ghrelin. | There is a well-documented relationship between short sleep duration and high body mass index (BMI). In the largest study, a survey on sleep duration and frequency of insomnia in more than 1.1 million participants, increasing BMI occurred for habitual sleep amounts below 7–8 hours [ 1 ]. A recent prospective study found an association between sleep curtailment and future weight gain [ 2 ]. The mechanism linking short sleep with weight gain is unknown, but Mignot and colleagues' study in this month's PLoS Medicine [ 3 ] adds to the growing evidence implicating leptin and ghrelin, the two key opposing hormones involved in appetite regulation. Hormones That Regulate Appetite Leptin, a peptide hormone secreted from white adipocytes, is implicated in the regulation of food intake and energy balance. The hormone acts on the central nervous system, in particular the hypothalamus, suppressing food intake and stimulating energy expenditure. Leptin production is primarily regulated by insulin-induced changes in adipocyte metabolism—its secretion levels correlate with adipocyte mass and lipid loads. The Land of Cockaigne, by Pieter Brueghel the Elder Leptin promotes inflammation. The hormone provides an interesting link between obesity and pathophysiological processes such as insulin resistance and atherosclerosis, and disorders such as autoimmune and cardiovascular diseases and the metabolic syndrome. Increased serum leptin levels in obesity and metabolic syndrome support the view that these disorders are in fact low-grade systemic inflammatory diseases, characterized by increased concentrations of proinflammatory cytokines like interleukin-6, tumor necrosis factor-α and leptin. Leptin's proinflammatory role suggests that it may link energy homeostasis to the immune system [ 4 , 5 ].Ghrelin is a peptide hormone that stimulates appetite, fat production, and body growth—leading to increased food intake and body weight. It is secreted into the circulation from the stomach, but is also synthesised in a number of other tissues, including the kidney, pituitary, and hypothalamus, suggesting that the hormone has both distant and local (endocrine and paracrine) effects. These effects include stimulating the secretion of growth hormone, prolactin, and adrenocorticotropic hormone, and a diabetogenic effect on carbohydrate metabolism [ 6 ]. The New Study In this study of 1,024 participants in the population-based Wisconsin Sleep Cohort Study [ 7 ], Mignot and colleagues found that in persons sleeping less than 8 hours, increased BMI was proportional to decreased sleep [ 3 ]. The researchers also found that shorter sleep times were associated with increased circulating ghrelin and decreased leptin, a hormonal pattern that is consistent with decreased energy expenditure and increased appetite and obesity. These findings confirm earlier clinical reports on the effects of sleep deprivation and extend them to include naturalistic sleep in a large, community-based population. The study provides an exciting addition to the growing literature showing relationships between sleep curtailment, metabolic hormones, and metabolic disorders (including obesity). The data have important implications for our understanding of obesity and related disorders in the general population, with one caveat: the study population was enriched with snorers, making the results less applicable to a general population. Mignot and colleagues' data are in accord with human and animal studies that show that experimental curtailment of sleep leads to lower levels of leptin [ 8 , 9 , 10 , 11 ] and increased ghrelin [ 12 ]. The new study therefore lends some support to the interpretation that reduced sleep levels cause the hormonal changes. But there is also evidence of opposite effects—that is, that administration of leptin [ 13 ] and ghrelin can alter sleep. Ghrelin administration has been found to increase non-REM sleep in humans and mice, possibly via its interactions with the sleep-inducing peptide growth hormone releasing hormone (GHRH). Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor, making it a candidate for an endogenous sleep-promoting factor [ 14 ]. Mignot and colleagues' study is congruent with the idea that inadequate sleep enhances ghrelin secretion, which in turn acts as an endogenous sleep factor in humans. This is an important new area of research that could conceivably lead to more physiological sleep aids than are currently available, with profound implications for improved public health. Overall, the available studies suggest the presence of reciprocal interactions between metabolic hormones and sleep, relationships that are poorly understood at present. Does sleep interact with metabolic hormones directly or via intervening factors such as sleep-related breathing disorders? Patients with obstructive sleep apnea have impaired sleep and higher ghrelin levels than BMI-matched controls, and treatment with continuous positive airway pressure reduces ghrelin to control levels [ 15 ]. Although sleep-disordered breathing (SDB) was measured in the present study, the SDB analyses were not shown, making it difficult to evaluate the influence of SDB on ghrelin and leptin in this population. There is a clear need for well-controlled, population-based studies that allow us to examine multiple relevant factors simultaneously. The present study highlights the importance of shortened sleep in relation to obesity, leptin, and ghrelin, a good start toward this goal. Sleep and Public Health Many other important questions remain, such as the roles that other hormones, cytokines, and SDB play in obesity. Many of the unanswered questions have important implications for public health. For example, diabetes, visceral obesity, hypertension, and hyperinsulinemia commonly aggregate together in large populations, and are considered a “metabolic syndrome” that has been linked to SDB [ 16 ] and to inflammatory disorders [ 17 ]. To what extent does long-term sleep curtailment contribute to these and related public health issues? The possible role of sleep restriction in autoimmune and inflammatory disorders is of particular interest in light of recent findings linking immune function with ghrelin and leptin. Ghrelin and its receptor are expressed in human T-lymphocytes, where they can inhibit cytokine activation, including interleukins, tumor necrosis factor-α and leptin [ 18 ]. Conversely, leptin stimulates cytokine activation and immune-cell proliferation, an effect that predisposes to inflammatory conditions [ 4 ]. Is it possible, then, that sleep-related changes in leptin and ghrelin influence the development of metabolic and immune disorders? Can biologically restorative sleep reverse disease progression? Can biologically restorative sleep be defined on the basis of metabolic hormone responses? Future research may answer some of these and other questions, further elucidating the role of sleep in public health. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535424.xml |
548140 | Inhibition of TNFalpha in vivo prevents hyperoxia-mediated activation of caspase 3 in type II cells | Background The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells). Methods Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits. Results In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells. Conclusion In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells. | Background Oxidative stress is an important factor of acute lung injury. Prolonged exposure to high concentrations of oxygen (hyperoxia) during mechanical ventilation represents a life-saving intervention for critically ill patients. However, it also induces oxidative stress to the lung. The development of therapeutic strategies, aiming to prevent lung injury depends on a better understanding of the underlying pathways of hyperoxia-induced pulmonary damage. Severe, long lasting hyperoxia causes an inflammatory reaction with an influx of inflammatory cells, cell proliferation and hypertrophy, an increase of cytokines, apoptotic activity and subsequent morphologic evidence of lung injury [ 1 ]. The first 24 to 48 hrs of oxygen exposure constitute the initiation phase of the pulmonary oxygen toxicity [ 1 ]. Even though no morphologic injury has been described during this phase, several changes occur due to the hyperoxic exposure. Highly reactive oxygen species are likely to cause lipid peroxidation, protein and DNA modification that will further cell injury [ 2 , 3 ]. On the other hand, antioxidant enzymes are also induced and may counteract the oxidative stress [ 4 - 6 ]. Perkowski et al. analysed more then 8700 genes during the early response (0 up to 48 hrs) to hyperoxia in total lung of mice. Out of 385 genes in the lung, 175 showed an increased and 210 a decreased expression [ 6 ]. These results indicate that the initiation phase of hyperoxic-induced lung injury already marks a very complex process that is still poorly understood. From previous investigations it may be concluded that in response to oxidative stress, the number of endothelial cells strongly decreases in the post-initiation phase, whereas epithelial cells seem to be relative resistant to oxidative stress [ 1 , 7 ]. In contrast, it has also been shown that in response to hyperoxic ventilation [ 8 ], emphysema [ 9 ], activation of the Fas/FasL system [ 10 ], exposure to donors of nitric oxide or hydrogen peroxide [ 11 ], hyperoxia and nitric oxide [ 12 ], respiratory distress syndrome [ 13 ], and hyperoxia-mediated increase of total lung p53 protein expression [ 14 ] alveolar type II cells (TIIcells) are severely damaged, culminating in apoptotic death. TIIcells are functionally highly important epithelial lung cells. They are responsible for the metabolism of alveolar surfactant, serve as progenitor cells of type I pneumocytes, and take part in the inflammatory response of the lung [ 15 - 17 ]. Thus, damage and apoptotic elimination of TIIcells will severely alter pulmonary function. Following the concept that hyperoxic lung injury is a continuous process, we assumed that appropriate metabolic changes of TIIcells start during the initiation phase. This would then, in response to longer lasting severe hyperoxia or an additional stress, merge in apoptosis in the post-initiation phase [ 18 ]. Factors which induce such pro-apoptotic sensitisation of TIIcells in the initiation phase are yet unknown. Elevation of tumour necrosis factor α (TNFα) represents one of the first pulmonary responses to hyperoxia. Pre-treatment of animals with antibodies directed against TNFα reduces hyperoxia-induced lung injury, strongly suggesting a causal relationship between TNFα and hyperoxic lung [ 19 ]. TNFα is a classic regulator of cell death by apoptosis or necrosis [ 20 , 21 ]. Cellular response to TNFα is mediated by TNF receptor type I and type II (TNFRI and TNFRII; [ 22 ]). Pryhuber et al. [ 23 ] studied the contribution of both, TNFRI and TNFRII to hyperoxia-induced lung injury and found that the average length of early survival under hyperoxic conditions is significantly improved in mice that lack the TNFRI (-/-), when compared with wild type or TNFRII (-/-) mice, respectively. However, the blockade of the TNFα receptor function does not protect against pulmonary inflammation and toxicity induced by prolonged hyperoxia [ 23 ]. In fact, during prolonged hyperoxia severe lung injury is most likely initiated by additional factors beside TNFα, as the inhibition of TNF receptors does not further affect oxygen-induced mortality [ 23 ]. Thus, TNFα and signal transduction via TNFRI seems to be responsible for metabolic changes that regulate the length of survival under short term hyperoxia. In this paper, we tested the hypothesis that TNFα activates caspases in the initiation phase of pulmonary oxygen toxicity in TIIcells without a significant increase in apoptosis. Since apoptosis is modulated by cell-matrix and cell-cell interactions in cultured TIIcells isolated from animals with acute lung injury [ 24 , 25 ]. we used freshly isolated TIIcells for our study. Materials and methods Hyperoxia Wistar rats (body wt 120 g), each in an individual plastic chamber were continuously gassed with 100 % oxygen for 48 hours. Water and food was available ad libitum . Preparation of the bronchoalveolar lavage, alveolar macrophages and TIIcells was carried out as previously described [ 26 ]. Immunohistochemistry Immunohistochemistry and microscopy were carried out as previously described [ 26 ]. The following antibodies were used: Rabbit polyclonal anti-rat TNFα antibody from Biosource Europe (Nivelles, Belgium), rabbit polyclonal anti TNFRI antibody raised against a recombinant peptide (amino acids 30–301) including the extracellular domain of TNFRI (Santa Cruz Biotechnology, Heidelberg, Germany), and anti-active caspase 3 polyclonal antibody was from Promega (Mannheim, Germany). Secondary antibodies conjugated with Alexa 499 and Alexa 594 were from Molecular Probes Europe BV (Leiden, Netherlands). Lung tissue sections were labelled with specific antibodies directed against TNFRI, TNFα, caspase 3, and p180 [ 27 ], an integral lamellar body-limiting membrane protein (clone 3C9, Covance/Berkeley Antibody, Richmond, CA, USA). For double staining, the labelled preparations were analysed using a confocal laser scanning microscope (CLSM, Leica Microsystems AG, Wetzlar, Germany), equipped with an argon/krypton laser. Images were taken using a 40 × NA 1.3 oil objective to fluorescent excitation and emission spectra for Alexa 488 (excitation 490 nm, emission 520 nm) and for Alexa 594 (excitation 541 nm, emission 572 nm). With the dual-channel system of the confocal microscope, dual-emission (535/590 nm) images were recorded simultaneously with a scanning speed at 16 s/frame (512 lines). Images were obtained and processed using TCS NT Version 1.5.451 (Leica Microsystems AG, Wetzlar, Germany). As controls, the tissue slides were incubated with the Alexa-labelled second antibodies only. No unspecific binding of the second antibodies occurred (results not shown). For threefold staining (Figure 6 ), fixed lung tissue and freshly isolated TIIcells were incubated in 0.01 M phosphate buffered saline containing 1% (w/v) BSA and 0.3% (w/v) Triton X-100 for 1 hr at room temperature (RT). Detection of lamellar bodies and active Caspase 3 was achieved by incubation with mab 3C9 (20 hrs at 4°C) followed by Alexa 488-labelled goat anti-mouse IgG (2 hrs at RT) and with anti-active caspase 3 followed by Alexa 594-labelled goat anti-rabbit IgG (2 hrs at RT), respectively. Nuclear DNA was stained with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes Europe BV, Leiden, Netherlands) for 20 minutes at RT. Figure 6 Hyperoxia activates caspase 3 in TIIcells. Rats were kept normoxic (control) or subjected to hyperoxia. Lung sections (A) and freshly isolated TIIcells (B) were immunohistochemically threefold stained as described in Materials and Methods . Cell nuclei stained light blue (DAPI). TIIcells are distinguishable by the close proximity of their nuclei to green stained lamellar bodies (A; arrows). Active caspase 3 appears red labelled and is predominantly found in the cytosol of TIIcells upon hyperoxic treatment of rats (A and B). Bar 10 μm; Laser scanning confocal microscopy was performed using a ZEISS LSM 510 system with Axiovert microscope (Carl Zeiss Jena GmbH, Jena, Germany) with 40×/1.3 Oil Dic or 63×/1.4 Oil Dic objective, equipped with an argon, helium/neon and violet laser set to 488, 543 and 405 nm, respectively. The multitrack standard FITC/Rhodamine/DAPI configuration was selected. Determination of apoptosis in lung tissue TUNEL reaction Sections of rat lung were prepared as described [ 26 ]. After deparaffinization and proteinase K-treatment, apoptotic cuts of chromatin DNA were specifically detected by nick end labelling of 3'-OH DNA ends with fluorescein-dUTP using terminal deoxynucleotidyl transferase. (MEBSTAIN Apoptosis Kit Direct, MBL, Naka-ku Nagoya, Japan). Sections were analyzed using a confocal laser scanning microscope as described above for double staining. Determination of apoptosis in freshly isolated TIIcells Cell Death Detection ELISA Cytoplasmic histone-DNA fragments were quantified using the Cell Death Detection ELISA (Roche, Mannheim, Germany). Flow cytometry We used the TACS™ Annexin V-FITC Detection kit (R&D Systems, Wiesbaden, Germany) to quantify the population of early and late apoptotic cells in percent of total cells. The tests were performed according to the protocols of the manufactures. Determination of caspase activities Activities of caspases 3, 8 and 9 were determined in the lysates of 8 × 10 6 TIIcells for each group and for each caspase with the Colorimetric assays from R&D Systems Inc. (Wiesbaden, Germany). When the pro-caspases had to be determined, an aliquot of the lysate (corresponding to 2 × 106 cells) was preincubated with 0.1 μg granzyme (Calbiochem, Bad Soden, Germany; dissolved in 5 μl 0.9% NaCl) for 30 min at 37°C. Determination of NF-κB activation Immunocytochemistry Activation of NF-κB was measured by immunocytochemistry using a monoclonal anti-NF-κB-antibody (MAB3026, Chemicon International, Temecula, USA), that recognises an epitope which includes the nuclear location signal of p65, the DNA binding subunit mainly responsible for the strong gene-inductory potential of NF-κB. Thus, only the activated form of NF-κB was measured. The semiquantitative estimation of NF-κB subunit by confocal microscopy was carried out as recently described in detail [ 28 ]. Immunoblotting Translocation of NF-κB to the nucleus was assessed as described by Li et al. by immunoblotting of nuclear extracts using a rabbit polyclonal antibody (biomol GmbH, Hamburg, Germany) directed against the p65-subunit [ 29 ]. Electrophoretic mobility shift assay (EMSA) was employed to detect the activated transcription factor NF-κB. Because this method is based on the binding of the transcription factors to their specific DNA recognition sequences, it is highly specific. Labelling of the NF-κB consensus oligonucleotide and handling of the assay were as described by the manufacturer (Gel Shift Assay Systems, Promega GmbH, Mannheim, Germany). Briefly, 50 micrograms of TIIcell nuclear extract were preincubated in reaction buffer for 10 minutes at RT. A [32P]-labelled oligonucleotide (Promega GmbH, Mannheim, Germany) which contains DNA binding sites for NF-κB transcription factors was then added to the reaction mixture and incubated for 20 minutes at RT. The complexes were separated on a 4% polyacrylamide gel that was dried and exposed to autoradiography. The specificity of the DNA-binding protein for the putative binding site was established by competition experiments using unlabelled NF-κB consensus oligonucleotide. Intratracheal application of anti-TNFα antibodies Wistar rats were lightly anaesthetised by inhalation of ether. The rats obtained intratracheally 50 μg goat IgG (PERBIO SCIENCE, Bonn, Germany) or 50 μg goat polyclonal anti-rat TNFα antibody (Santa Cruz Biotechnology, Heidelberg, Germany) per animal, respectively. Thereafter the rats were kept at hyperoxic conditions as described in hyperoxia. After 48 hrs, the TIIcells were isolated and TNFRI expression and caspase-3 activity were determined as described above. Other methods For the determination of the TNFα concentration in TIIcells, macrophages, plasma or cell-free bronchoalveolar lavage, we used a commercially available ELISA kit from Biosource (Ratingen, Germany). The determination of different apoptotic markers [ 18 ], Western blot analysis [ 17 ], mRNA isolation and Real-time quantitative PCR reaction for the determination of the expression of mRNAs in TIIcells was described in detail previously [ 26 ]. GSH, GSSG and GSH-reductase were determined by HPLC with subsequent fluorescence detection as previously described [ 17 ]. Statistical analysis Differences between two groups were assessed using the Student's t-test. Probability values < 0.05 (two-tailed) were considered significant (see legends of Tables and Figures). Results Hyperoxia-induced changes in lung tissue To test the general usefulness of our hypothesis, we first characterised the oxygen-induced changes in lung-tissue-sections. Immunohistochemically, we observed a clear increase in TNFα, TNFRI, and caspase 3 activities in lung tissue of hyperoxic rats in relation to normoxic animals in the initiation phase (Figure 1 ). Albeit the increase of these pro-apoptotic parameters in lung tissue corroborated our hypothesis, this approach does not allow to identify the participating cell types. Furthermore, we tested lung sections for DNA-degradation products using the TUNEL reaction (Figure 2 ). Here, TIIcells are distinguishable from other cells of the lung by their content of lamellar bodies. Lamellar bodies were immunohistochemically labelled in lung sections with an antibody directed against the 180-kDa lamellar body-limiting membrane protein (red, Figure 2 ). Upon hyperoxia, we found sporadically a fragmentation of DNA (green, Figure 2 ), but no co-localisation of the 180-kDa lamellar body-limiting membrane protein and DNA-fragments. The intensity of red lamellar body stain increased in lung sections from hyperoxic rats. This is in good accordance with previously published electron micrographs showing swollen and deformed lamellar bodies after hyperoxia [ 30 ]. From these results, we conclude in agreement with the literature [ 1 , 7 , 25 ]. that sublethal hyperoxia of rats did not induce apoptosis in TIIcells in vivo ; at least not in the initiation phase. Figure 1 Hyperoxia increases the expression of TNFRI, TNFα and caspase 3 in vivo. Lung sections of normoxic (control) and hyperoxic rats were prepared as described in Materials and Methods and were labelled by single immunofluorescence with antibodies directed against TNFRI, TNFα or active caspase 3 Figure 2 No significant apoptosis was found in TIIcells upon hyperoxia in vivo for 48 hrs. Lung tissue of normoxic rats (left) and of hyperoxic rats (right) were tested for DNA fragmentation by TUNEL reaction as described in Materials and Methods . The positive TUNEL reaction is represented by green fluorescence. The presence of lamellar bodies is indicated by red fluorescence. Pseudo-colour blue was used to highlight the contours of lung tissue. Bar: 25 μm. The results indicate that apoptotic parameters as are TNFα content, TNFRI expression and caspase-3 activity increase in lung tissue during the initiation phase but do not induce TIIcell apoptosis in vivo . Following our hypothesis, we examined whether TIIcells undergo oxidative stress at our conditions, and whether an increase of TNFα, TNFRI expression, and caspase-3 activity appears in isolated TIIcells. Hyperoxia-induced oxidative stress of freshly isolated TIIcells The determination of cellular GSH, oxidised GSH (GSSG), and the activity of the GSH-reductase showed the oxidative burdening of TIIcells. In response to hyperoxia, the GSSG content significantly increased and the GSH reductase activity significantly decreased (Table 1 ). The GSH content in freshly isolated TIIcells has rarely been determined. In our TIIcell population, the GSH content differs in relation to previously published data of freshly isolated TIIcells from rat [ 31 ] and rabbit [ 32 ] by the factor of about 2 and 4, respectively. These differences might be explained by different methods of TIIcell isolation and by species specificity. Table 1 Effect of hyperoxia on GSH reductase activity, and on GSH and GSSG content in TIIcells Control Hyperoxia GSH reductase (% of control) 100 76 ± 10* GSH (μmol/mg protein) 0.022 ± 0.015 0.020 ± 0.008 GSSG (μmol/mg protein) 0.005 ± 0.0015 0.013 ± 0.001* GSH/(GSH+GSSG) (ratio) 0.81 0.61 Activity of glutathione (GSH) reductase and concentration of GSH and GSSG were determined in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) as described in Materials and Methods . Values are means ± standard deviation of n = 3 independent experiments. Asterisk indicates a significant difference to control ( p < 0.05). The ratio GSH/(GSH+GSSG) is one of the most sensitive parameters to describe oxidative burdening. Our values are comparable to the values found by van Klaveren et al. in freshly isolated type II cells (0.816 versus 0.815) [ 30 ]. This ratio decreased in response to hyperoxia in vitro to 0.74 [ 31 ] and in our in vivo approach to 0.61 (Table 1 ). With respect to the published data, we conclude that our treatment induced oxidative burdening in freshly isolated type II cells. Hyperoxia activates NF-κB NF-κB activation has been described as an indicator of oxidative stress [ 33 , 34 ]. In response to sublethal hyperoxia, the content of activated NF-κB in TIIcells clearly increased (Figure 3A ). The picture shows that the larger portion of activated NF-κB seems to be localised in cytosol. The semiquantitative determination of activated NF-κB showed a significant increase (control: 12.7 ± 9.0; hyperoxia: 59.5 ± 11.6; n = 12; p < 0.01) in the nucleus of TIIcells in response to hyperoxia. In the cytosol, there was also an increase of activated NF-κB (1.6-fold), however, this difference curtly fails the level of significance. Additionally, we detected a translocation of the NF-κB-subunit p65 into the nuclear protein fraction as estimated by Western blot analysis (Figure 3B ). Hyperoxia increased the content of the p65-subunit in the nuclear protein fraction of TIIcells 1.68-fold (SD 0.58, n = 4) compared to normoxic control, but the difference did not reach significance. Whether the immunoreactive band above the p65-subunit in the Western blot of the hyperoxic group is non-specific or a possible post-translational modification can not be decided. In Figure 3C we confirm the hyperoxia induced activation of NF-κB by EMSA of the nuclear protein fractions of TIIcells freshly isolated from control (lanes 1, 2) and hyperoxic (lanes 3, 4) rats. Addition of unlabelled specific oligonucleotide clearly competes with the [32P]-labelled probe confirming the specificity of the bands. Figure 3 Activation of NF-κB in TIIcells and its enrichment in the nuclear protein fraction after hyperoxic treatment of rats. Freshly isolated TIIcells from normoxic (control) and hyperoxic rats were prepared for immunocytochemistry, SDS-PAGE and immunoblotting as described in Materials and Methods . A, activation of NF-κB was measured by immunocytochemistry using a monoclonal anti-NF-κB-antibody overlapping the nuclear localisation signal of the p65 subunit in the NF-κB heterodimer. Activated NF-κB in the nucleus and cytosol was quantified as described recently in detail [28]. The signal of activated NF-κB in the nucleus increased 4.7 fold in respose to hyperoxia (n = 12; p < 0.05). Bar: 10 μm. B, the nuclear protein fraction [52] of TIIcells was subjected to SDS-PAGE and immunoblotting. The p65 subunit of NF-κB was visualised using a rabbit polyclonal antibody, and its expression was densitometrically estimated. Values of n = 4 independent experiments are given as mean ± SD in arbitrary units (control = 1). C, Electrophoretic mobility shift assay for NF-κB in freshly isolated TIIcells from normoxic (lanes 1, 2) and hyperoxic (lanes 3, 4) rats. Signal competition upon addition of unlabelled oligonucleotide (lanes 2, 4). Hyperoxia increases synthesis and secretion of TNFα by TIIcells The concentration of TNFα significantly increased in response to hyperoxia of rats in plasma, alveolar fluid, lung macrophages and TIIcells (Table 2 ) as determined by ELISA. Flow cytometric analysis of the TNFα content in freshly isolated TIIcell preparations from control and hyperoxic rats confirmed the increase of the TNFα concentration in macrophages and TIIcells. In response to hyperoxia, TNFα increased in TIIcells 1.45-fold and in macrophages 1.87-fold compared to control. In TIIcells, TNFα seems to be localised in lamellar bodies (Figure 4 ), whereas cytoplasmic caspase 3 showed no co-localisation with lamellar bodies as expected. The latter result attaches value to the histochemically detected localisation of TNFα in lamellar bodies, because it is unlikely an artefact. The spontaneous secretion of TNFα by TIIcells significantly increased in response to hyperoxia (Table 2 ). Table 2 Effect of hyperoxia on the TNFα content in different specimen from rat Control Hyperoxia Plasma (pg/ml) 106 ± 31 149 ± 11 Macrophages (ng/mg protein) 11.4 ± 3.7 19.4 ± 2.8* TIIcells (ng/mg protein; n = 6) 18.5 ± 2.1 27.2 ± 6.7* Bronchoalveolar lavage (pg/ml) 109 ± 1 172 ± 38* Spontaneous secretion of TNFα by TIIcells (ng × mg cell protein -1 × hr -1 ) 6.3 ± 1.3 21.2 ± 7.5* Concentration of TNFα was determined in plasma, macrophages, TIIcells and bronchoalveolar lavage of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) by ELISA as described in Materials and Methods . Spontaneous secretion of TNFα was measured in cell-free supernatant upon incubation of freshly isolated TIIcells in DMEM for 30 min at 37°C. TNFα concentrations are given as means with standard deviation of n = 3 independent experiments unless stated otherwise. Asterisk indicates a significant difference to control ( p < 0.05). Figure 4 TNFα is localised in lamellar bodies and caspase 3 in the cytosol of TIIcells. After hyperoxia, rat lungs were fixed and the sections were immunohistochemically double labelled as described in Materials and Methods . A: bar 50 μm; B: higher magnification of the indicated area of A (arrow); bar 10 μm. By confocal microscopy, TIIcells were identified by the green labelling of lamellar bodies (see Methods). The red labelled TNFα appears yellow (arrow in B) when co-localised in lamellar bodies as shown in two TIIcells in B. Hyperoxia induces the expression of TNFRI and activates caspases in TIIcells Hyperoxia induced an significant increase of TNFRI on TIIcells (4.9-fold ± 2.7, n = 6), whereas the expression of Fas, a member of the same receptor family, did not change (Figure 5 ). TNFRI-mediated action of TNFα depends on the so called "death domain" representing a part of the intracellular segment of the receptor protein responsible for the activation of pro-caspase 8. Caspase 8 in turn can activate pro-caspase 3, a feature previously reviewed [ 35 - 37 ]. We show in situ that the activation of caspase 3 actually occurs in TIIcells and that caspase activation as a response to an unspecific stress, e.g. isolation, can be excluded (Figure 6 ). This staining technique excepts cytoplasm and membranes, thus, cells can hardly be delimited. However, TIIcells are identifiable by the immediate proximity of their nuclei to lamellar bodies (green). Figure 5 Hyperoxia increases the expression of TNFRI but not of Fas in TIIcells. For the expression analysis of TNFRI and Fas the membrane fraction of freshly isolated TIIcells was prepared. TNFRI (A) and Fas (B) were visualised by Western blot technique, and their expression was densitometrically determined. Values of n = 6 independent experiments are given as means ± SD in arbitrary units (control = 1). Asterisk indicates a significant difference to control ( p < 0.05). As shown in Table 3 , in TIIcells the activity of caspase 8 and 3 increased in response to hyperoxia, whereas the activity of caspase 9 did not change. Pre-incubation with granzyme activated pro-caspases and resulted in an increase of caspase 8 and -3 activities in control and hyperoxic TIIcells (Table 4 ). However, the activation in control cells clearly exceed that in hyperoxic TIIcells, indicating that pro-caspases were already, at least in part, activated in response to hyperoxia. Table 3 Effect of hyperoxia on the activity of caspases in TIIcells Control Hyperoxia Caspase 8 (n = 6) 100 143 ± 14* Caspase 3 (n = 6) 100 168 ± 23* Caspase 9 (n = 3) 100 108 ± 11 Caspase activity in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) was determined colorimetrically as described in Materials and Methods . Values are means ± S.D. given in arbitrary units (control = 100). Asterisk indicates a significant difference to control ( p < 0.05). Table 4 Effect of granzyme-treatment on the activity of caspases in TIIcells from normoxic and hyperoxic rats Control Hyperoxia Granzyme - + - + Caspase 8 100 155 ± 7* 100 110 ± 4* Caspase 3 100 216 ± 14* 100 155 ± 10* Caspase activity in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) was determined colorimetrically as described in Materials and Methods . In some experiments, cell lysates were incubated in the presence of granzyme prior to determination of caspase activity (see Methods ). Values are means ± s. d. given in arbitrary units (values without granzyme = 100) of n = 3 independent experiments. Asterisk indicates a significant difference between granzyme-treated and untreated samples ( p < 0.05). Anti-TNFα in vivo prevents hyperoxia-driven increase in TNFRI and in active caspase 3 In order to demonstrate the causality between hyperoxia and TNFRI-mediated activation of caspases, we attempted to bind TNFα by anti-TNFα antibodies. Table 5 shows that a single intratracheal application of anti-TNFα antibodies immediately preceding hyperoxic treatment prevents the hyperoxic-induced increase of TNFRI expression and caspase-3 activity in freshly isolated TIIcells. These results indicate that both TNFRI expression and caspase-3 activation were induced by TNFα. This corroborates the concept that in a cascade starting by the TNFα/TNFRI-interaction caspase 8 is activated which in turn activates caspase 3. Table 5 Effect of intratracheal application of anti-TNFα antibodies on hyperoxic induced parameters of TIIcells Hyperoxia without with anti-TNFα antibody TNFRI 100 26 ± 19* Caspase 3 activity 100 64 ± 5* Rats obtained intratracheal 50 μg goat-IgG (without) or 50 μg goat polyclonal anti-TNFα antibodies (with anti-TNFα antibody) preceding hyperoxic treatment. Hyperoxia of rats was carried out as described in Material and Methods . Values are means ± SD of n = 3 independent experiments given in percent (hyperoxia without anti-TNFα antibody = 100). Determination of TNFRI (Western blot) and of caspase 3 activity (colorimetrically) was as described in Material and Methods .). Asterisk indicates a significant difference compared to control ( p < 0.05). Hyperoxia upregulates genes of TNFRI, TNFα and caspases 3 and 8 The content of individual mRNAs in TIIcells was determined by Real-time PCR. In agreement with microarray analysis in total lung of mice [ 6 ] we found that the amount of GAPDH mRNA did not change in the first 48 hrs of hyperoxia (results not shown). Therefore, GAPDH was used as a house keeping gene. In contrast, Ho et al. found a small but significant increase of GAPDH mRNA in lung tissue [ 5 ]. This difference may be caused by different base material (TIIcells versus lung tissue) and methodical differences (Taqman versus densitometry of autoradiographs). In response to sublethal hyperoxia the mRNA content of TNFα, TNFRI and caspases increased (Table 6 ). The increment of caspase-8 mRNA was not significant, but even a ΔΔc t of -1.15 indicates a 2.2-fold increase of mRNA content. Table 6 Effect of hyperoxia on the mRNA content of TNFα, TNFRI, and caspases in TIIcells number of cycles (Δc t ) increase of mRNA Control Hyperoxia ΔΔc t -fold TNFα 3.66 ± 0.66 0.93 ± 0.30* -2.73 6.6 TNFRI 5.02 ± 0.19 4.44 ± 0.11* -0.58 1.5 Caspase 8 10.3 ± 0.89 9.15 ± 0.35 -1.15 2.2 Caspase 3 5.33 ± 0.25 4.29 ± 0.48* -1.04 2.1 The mRNA of three animals per group was isolated and combined. The content of specific mRNA was determined using Real time-PCR. In parallel, GAPDH was determined in each run as internal standard. The values are given as number of GAPDH-corrected cycle (Δc t ) ± SD of n = 3 determinations. ΔΔc t is the difference of the number of cycles between control and hyperoxia; a decrease of Δc t indicates an increase of the mRNA, also given as a factor of increase. Asterisk indicates a significant difference to control ( p < 0.05). Hyperoxia of rats does not induce apoptosis in freshly isolated TIIcells As mentioned above in this section, no increase of apoptosis was detected in TIIcells in response to sublethal hyperoxia by immunohistochemistry (Figure 2 ). To confirm this result, we analyzed biochemical parameters of apoptosis in freshly isolated TIIcells. In agreement with our immunohistochemical results, sublethal hyperoxia of rats did not increase apoptosis in freshly isolated TIIcells (Table 7 ). Hyperoxia was without effect on anti-apoptotic Bcl-2, and cytosolic cytochrome C, although the pro-apoptotic Bax increased and the mitochondrial transmembrane potential slightly decreased (Table 7 ). In accordance with these results, the activity of caspase 9 did not change. Therefore, activation of caspase 3 seems to be catalysed by caspase 8. Table 7 Effect of hyperoxia on apoptotic parameters in TIIcells Control Hyperoxia Bcl-2 (n = 5) 100 96 ± 8 Bax (n = 5) 100 134 ± 28* Cytochrome c (n = 5) 100 102 ± 15 Mitochondrial membrane potential 100 81 ± 8* Early apoptotic TIIcells 100 101 ± 26 Late apoptotic TIIcells 100 94 ± 22 Cell death detection ELISA 100 104 ± 13 Several apoptotic parameters were determined in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) as described in Materials and Methods . Values are means ± S.D. given in arbitrary units (control = 100) of n = 3 independent experiments unless stated otherwise. Asterisk indicates a significant difference to control ( p < 0.05). Hyperoxia of rats followed by normoxia reduced caspase 3 activity but did not increase apoptosis in TIIcells After hyperoxia for 48 hrs, we detected an activation of caspase 8 and caspase 3. Much to our surprise, we found no increase in apoptosis of TIIcells although the activity of these caspases increased. However, apoptosis might occur later than 48 hrs and will not necessarily arise concomitantly with caspase activation. Thus, following hyperoxia the animals were kept for 24 and 48 hrs under normoxic conditions to test TIIcells for appearance of apoptosis at a later time point. During normoxia, caspase 3 activity gradually decreases compared to control, whereas apoptosis did not change significantly as detected by cell death detection ELISA and by the number of early and late apoptotic cells (Table 8 ). Table 8 Apoptotic parameters upon sublethal hyperoxia of rats followed by normoxia Hyperoxia followed by normoxia for 48 hrs hyperoxia 24 hrs normoxia 48 hrs normoxia Caspase 3 activity 171 ± 18* 97 ± 16 73 ± 29 Early apoptotic cells 102 ± 13 95 ± 14 109 ± 26 Late apoptotic cells 106 ± 29 125 ± 25 85 ± 17 Cell death detection ELISA 97 ± 23 104 ± 6 95 ± 14 Activity of caspase 3 and parameters of apoptosis detected in freshly prepared TIIcells of rats exposed to oxygen for 48 hrs (hyperoxia) followed by normoxia for 24 or 48 hrs as described in Materials and Methods. Values are means ± S.D. given in arbitrary units (normoxic control = 100) of n = 3 independent experiments. Asterisk indicates a significant difference compared to control ( p < 0.05) Discussion Short-time hyperoxia, as used in this experiments represents the initiation phase of lung injury [ 1 ]. We started our investigations with the aim to characterise metabolic changes in TIIcells taking place in this phase. On the one hand, these changes should be less complex than in post-initiation phases. On the other hand, therapeutic interventions to avoid or minimise hyperoxia-induced lung injury should focus in particular on this phase, because morphologic injury of lung tissue, inflammation, and death of lung cells originate from here. Furthermore, the metabolic changes in the initiation phase may, at least in part, be still reversible. For the first time, we show that sublethal hyperoxia causes not only an increase in TNFα concentration in lung tissue (as previously published [ 38 , 39 ]), but also in freshly isolated TIIcells and that this increase is combined with an enhanced expression of TNFRI and an activation of caspase 3. It has been widely assumed that macrophages are the source of TNFα in alveolar fluid [ 40 ]. Our findings provide evidence that hyperoxic treatment of rats provokes a rise in cellular TNFα not only in alveolar macrophages [ 41 ]. We show that the TNFα-gene is up-regulated in TIIcells; in parallel, the TNFα-protein content increased. To the best of our knowledge, our data suggests for the first time that TNFα is localised in lamellar bodies. In the light of this assumption, the secretion of TNFα as a constituent of the lamellar bodies by TIIcells might represent a significant contribution to the increased TNFα-content in alveolar fluid. Whether the small increase of the TNFα concentration in plasma observed by us indicates a beginning systemic inflammation or rather reflects a transfer of TNFα from the alveolar space to plasma, remains open (Table 2 ). Cellular effects of TNFα are mediated mainly by its specific receptor, TNFRI. In agreement with our hypothesis, the expression of TNFRI in TIIcells is up-regulated on mRNA and protein level in response to sublethal hyperoxia, while the Fas-expression did not change, although both receptors belong to the same family (Figure 5 ). It may be speculated that a parallel increase of effector and specific receptor always occurs when the cellular metabolism in TIIcells can be affected by an autocrine mechanism. The increase of Fas/Fas-Ligand might be characteristic in post-initiation phases. The intracellular part of the transmembrane TNFRI protein contains the "death domain" which is responsible for the activation of caspase 8. This can trigger the path to the final part of apoptosis via activation of caspase 3 [ 36 ]. In line with this, we found enhanced levels of caspases 8 and 3, yet caspase 9 was not activated. The latter result is corroborated by the absence of an increased mitochondrial cytochrome C release. The data presented here demonstrate that sublethal hyperoxia of rats did not induce apoptosis in TIIcells despite of the increase in TNFα content, TNFRI expression, and activation of caspase 3. To check whether this pro-apoptotic state in TIIcells is indeed triggered by TNFα and whether it may be reversible, anti-TNFα antibodies were administered intratracheally prior to hyperoxic exposure. We show that this treatment completely prevented hyperoxic-induced increase of TNFRI expression and caspase 3 activation. It is a widely accepted concept that activation of caspase 3 marks the "point of no return" in the pathway of apoptotic death of mammalian cells. However, we found that neither in lung tissue nor in freshly isolated TIIcells apoptosis occurs in response to sublethal hyperoxia despite the significant activation of caspases 8 and 3. In other words, the increase of caspase 8 and 3 in response to sublethal hyperoxia did not mark the "point of no return" in TIIcells. This interpretation of our results is strongly corroborated by Perfettini and Kroemer [ 37 ]. They summarised that caspase inhibition does not avoid but actually encourage death in TNF induced shock, indicating that caspase activation is not basically synonymous with apoptotic cell death. It could be argued that no apoptosis was found in freshly isolated TIIcells because the increase of caspase 3 activity and the increase of apoptotic parameters does not occur concomitantly. However, we found no increase in apoptosis of TIIcells in a normoxic period of 24 and 48 hrs directly succeeding the hyperoxic treatment of animals, whereas the caspase 3 activity gradually decreased. These results indicate that caspase 3 activation in response to sublethal hyperoxia is reversible and does not kill TIIcells essentially. The reason why the activation of caspases did not induce apoptosis of TIIcells is not clear. On the one hand, activation of NF-κB has often been implicated as an anti-apoptotic event [ 42 - 45 ], and TNFα induces also the expression of anti-apoptotic TNFα-receptor-associated-factors in lung cells and might inhibit by this way TNFα-induced cell death or apoptosis [ 46 ]. Therefore, it may be assumed that both NF-κB activation and expression of anti-apoptotic TNFα-receptor-associated-factors arrest the hyperoxia-induced metabolic changes of TIIcells in the pro-apoptotic state. On the other hand, it can not be excluded that the extend of caspase 3 activation (1.68-fold compared to control) is not high enough to induce apoptosis of TIIcells although a 1.8-fold increase of caspase 3 is combined with apoptosis in total lung of a hyperoxia/pneumonia model [ 47 ]. We hypothesise that caspase 3 activation is then followed by apoptosis, when its activation occurs via strong mitochondrial damage resulting in cytochrome c release and caspase 9 activation in post-initiation phases of pulmonary oxygen toxicity. This concept is supported by recent findings that mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury [ 48 ]. In fact, the mitochondrial membrane potential and Bax significantly changed in TIIcells in the initiation phase of hyperoxic lung injury, but without cytochrome c release or activation of caspase 9. Recently, it has been shown that in response to severe hyperoxia of mice apoptosis and necrosis contribute to an extensive cell death; p53, bax, bcl-x, and Fas increased in mRNA and protein level, but the activity of caspase 3 and caspase 1 did not change [ 49 ]. This independence of apoptosis from caspase activities in the lung has also been shown in freshly isolated TIIcells. Previously, we showed that an increase of TIIcell-apoptosis in response to vitamin E deficiency of rats is independent of caspase activation [ 18 ]. Furthermore, using p53-deficient and Fas-null mice, Barazzone et al. [ 49 ] showed that Fas and p53 activation exhibits no linkage to lung injury in response to severe hyperoxia of mice. Contrariwise, it has also been shown that Fas activation in vitro [ 50 ] and in vivo results in TIIcell apoptosis and lung inflammation [ 10 , 51 ]. These results confirm that hyperoxic-induced lung injury is multifactorial, and that the elimination of one factor in the network of factors activated in the post-initiation phases often can not avoid lung injury in response to severe hyperoxia. In summary, TIIcells were not killed in the initiation phase of pulmonary oxygen toxicity. More precisely, its pro-apoptotic sensitisation is the background that, together with an additional stress factor, hyperoxia causes lung injury probably by apoptotic elimination of TIIcells. In agreement with this idea, we showed that the combination of the stress factors hyperoxia and vitamin E deficiency increases TIIcell apoptosis [ 18 ]. Taking into account that premature neonates exhibit vitamin E deficiency, it is consequential that ventilation of premature neonates suffering from respiratory distress syndrome with high levels of inspired oxygen amplifies lung injury, which is associated with TIIcell apoptosis [ 13 ]. Conclusions In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFα effect in vivo by anti-TNFα antibodies prevents the pro-apoptotic sensitisation of TIIcells. List of abbreviations DAPI – 4',6-diamidino-2-phenylindole; EMSA – electrophoretic mobility shift assay; GSH – gluthatione; GSSG – oxidised gluthatione; NF-κB – nuclear factor-κB; TIIcell – type II pneumocyte; TNFα – tumour necrosis factor α; TNFR – TNFα receptor; TUNEL – terminal transferase dUTP nick end labelling Authors' contributions HW carried out the immunohistochemistry, CS performed the mRNA analysis, AT carried out the protein measurements and participated in data analysis, MR applicated anti TNFα antibodies intratracheally, FS participated in the mRNA analysis and the design of the study, FG and BR conceived of the study, participated in its design and co-ordination, analysed the data and wrote the manuscript. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548140.xml |
544550 | Correction: Regulation of Muscle Fiber Type and Running Endurance by PPARδ | null | In PLoS Biology , volume 2, issue 10: Regulation of Muscle Fiber Type and Running Endurance by PPARδ. Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, et al. 10.1371/journal.pbio.0020294 The following competing interest should have been indicated in the above research article. R. M. Evans wishes to acknowledge a consulting relationship with Ligand Pharmaceuticals. Under a licensing agreement with GlaxoSmithKline, Ligand receives milestone payments on the development of GW501516, a PPARδ-specific drug used in this study. For clarification, no materials or support were received from either company, and no agreements were in place concerning the execution or publication of this work. Published January 18, 2005 | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544550.xml |
553992 | Astrocyte production of the chemokine macrophage inflammatory protein-2 is inhibited by the spice principle curcumin at the level of gene transcription | Background In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2) is thought to play a pivotal role in the induction and perpetuation of inflammation in the central nervous system (CNS). The origin of MIP-2 in inflammatory disorders of the brain has not been fully defined but astrocytes appear to be a dominant source of this chemokine. Curcumin is a spice principle in, and constitutes approximately 4 percent of, turmeric. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. To examine the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its known anti-oxidant activities, we studied the effect of this spice principle on the synthesis of MIP-2 by astrocytes. Methods Primary astrocytes were prepared from neonatal brains of CBA/CaJ mice. The cells were stimulated with lipopolysaccharide in the presence or absence of various amount of curcumin or epigallocatechin gallate. MIP-2 mRNA was analyzed using semi-quantitative PCR and MIP-2 protein production in the culture supernatants was quantified by ELISA. Astrocytes were transfected with a MIP-2 promoter construct, pGL3-MIP-2, and stimulated with lipopolysaccharide in the presence or absence of curcumin. Results The induction of MIP-2 gene expression and the production of MIP-2 protein were inhibited by curcumin. Curcumin also inhibited lipopolysaccharide-induced transcription of the MIP-2 promoter reporter gene construct in primary astrocytes. However MIP-2 gene induction by lipopolysaccharide was not inhibited by another anti-oxidant, epigallocatechin gallate. Conclusion Our results indicate that curcumin potently inhibits MIP-2 production at the level of gene transcription and offer further support for its potential use in the treatment of inflammatory conditions of the CNS. | Background Curcumin (1,7-Bis 94-hydroxy-3-methoxyphenyl)-1,6 heptadiene-3, 5-di-one) is a spice principle in and constitutes approximately 4% of turmeric and is responsible for curry's characteristic yellow color. As is true of other naturally occurring polyphenolic compounds, such as caffeic acid phenyl ester, rosmaric acid and resveratrol, curcumin possesses antioxidant properties which may reduce the production of free radicals and improve cell viability under conditions of enhanced oxidative stress[ 1 , 2 ]. Curcumin also has anti-inflammatory properties which include the capacity to inhibit 5- and 8-lipoxygenases and cyclooxygenases[ 3 , 4 ], is chemopreventive as evidenced by its capacity to abrogate 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced DNA synthesis and tumor promotion in mouse skin[ 5 ], antiproliferative as shown by its suppressive effect on the growth of C6 glioma cells[ 6 ], and anti-metastatic as suggested by its ability to inhibit angiogenesis in vivo [ 7 ]. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation such as Alzheimer's disease[ 8 ]. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. An active role for chemokines has been demonstrated in the pathogenesis of a variety of central nervous system (CNS) disorders accompanied by inflammation. In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis (EAE) and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2) appears to play a pivotal role in the induction and perpetuation of inflammation in the brain[ 9 , 10 ]. In EAE, for example, elevated levels of MIP-2 mRNA and protein preceded infiltration of the CNS by polymorphonuclear leukocytes (PMNs). Similarly, in traumatic brain injury, the kinetics of MIP-2 expression paralleled the recruitment of neutrophils to the inflammatory site[ 10 ] and, in experimental bacterial meningitis, neutralization of MIP-2 with a monoclonal antibody attenuated infiltration of the CNS with PMNs[ 11 ]. The origin of MIP-2 in inflammatory CNS disorders has not been fully defined, but in EAE astrocytes, appear to be the dominant source of this chemokine[ 9 ] and are likely to contribute significantly to MIP-2 production in other neuropathological states as well. To explore the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its antioxidant and known anti-inflammatory activities, we examined the effect of this spice principle on the synthesis of MIP-2 by astrocytes. Our results indicate that curcumin potently inhibits MIP-2 production at the level of gene transcription and offer further support for its potential use in the treatment of inflammatory conditions of the CNS. Methods Mice Six to eight-week-old CBA/CaJ mice were purchased from Jackson Laboratories (Bar Harbor, ME) and bred in our animal facility. Materials Curcumin, epigallocatechin (EGCG) and E. coli lipopolysaccharide (LPS; O55B1) were purchased from Sigma, (St Louis, MO). Rabbit anti-cow glial fibrillary acidic protein polyclonal antibody was obtained from Dako Corp. (Carpinteria, CA). Preparation and culture of astrocytes: Astrocytes were prepared from the brains of neonatal (3 to 7-day-old) CBA/CaJ mice by a modification of the method of Pousset et al[ 12 ]. Briefly, four brains were combined, homogenized in 0.25% trypsin through a sterile screen (pore size; 100 μM), incubated for 5 min at 37°C and centrifuged at 400 × g . The pellet was suspended in Hank's Buffered Salt Solution (HBSS) and debris was removed by gravity sedimentation on ice for 3 min. The supernatant was collected, centrifuged and the pellet was washed twice with culture medium consisting of DMEM containing 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 1 mM L-glutamate and penicillin/streptomycin (Gibco BRL, Grand Island, NY). The cells were plated on 35 mm dishes and cultured at 37°C in a humidified atmosphere contain 5% C02. After 16 hours, plates were washed to remove non-adherent cells and debris. For experiments in which mRNA or MIP-2 protein were quantified, adherent cells were cultured until they reached confluence. For transfection experiments, adherent cells were cultured until they were nearly confluent. Medium was refreshed in all astrocyte cultures every 2–3 days. The preparations were >98% glial fibrillary acidic protein positive, as measured by flow cytometric analyses using a EPICS XL flow cytometer[ 13 ]. Cell viability determination: The effect of curcumin on the viability of astrocytes was assessed by measuring cytosolic lactate dehydrogenase (LDH) leakage into the media as detailed earlier[ 14 ]. Briefly, astrocytes were incubated with curcumin (10- 4 M to 10- 6 M) for up to 48 hours, the supernatants were then harvested and LDH was measured by colorimetric assay using a kit from Sigma diagnostics. mRNA and protein analyses: Confluent cultures of astrocytes were incubated with LPS (10 ηg to 5 μg/ml) for varying periods of time in the presence or absence of curcumin (10- 4 M to10- 7 M). After 4 hours of culture, cells were harvested and mRNA was isolated as previously reported[ 14 ]. MIP-2 mRNA levels were determined using semi-quantitative polymerase chain amplification (PCR) as described earlier[ 14 ] using the primers: 5'-TGCCGGCTCCTCAGTGCT-3' (forward) and 5'-GCCTTGCCTTTGTTCAGTATCTTTTG-3' (backward). In other experiments, the effect of EGCG on induced MIP-2 mRNA production was determined by culturing astrocytes with LPS in the presence or absence of varying doses of the catechin (10- 3 M to 10- 4 M). To assess the effect of curcumin on MIP-2 protein production, astrocytes were cultured with LPS in the presence or absence of curcumin (10- 5 M) for 16 hours. Supernatants were then harvested and MIP-2 levels were determined by enzyme linked immunosorbant assay (ELISA; R&D systems, Minneapolis, MN). Preparation of the reporter gene, pGL3-MIP-2: A 537 base pair MIP-2 fragment was prepared by amplifying rat genomic (kidney) DNA using the primers: 5'GCCCACCGAGTCTCTGTTTC3' (forward) and 5'GTTGGTGGCCAGCAGGAGGA3' (backward), then digesting with Rsa I/Nco I. The fragment, which corresponded to base pairs -539 to -2, relative to adenine (assigned +1) in the translation initiation codon of the MIP-2 gene (accession number AJ49888), was ligated to a Sma I/Nco I digested, promoterless luciferase reporter vector, pGL3-Basic (Promega, Madison, WI). The direction of the insert was confirmed by restriction endonuclease digestion and its fidelity determined by sequence analyses as previously described[ 15 ]. The MIP-2 promoter-reporter gene construct, pGL3-MIP-2 is shown in Figure 1 . Figure 1 PGL3-MIP-2. A 537 bp fragment, which corresponds to base-pairs -539 to -2 (relative to adenine [assigned +1] in the translation initiation codon of the MIP-2 gene) was generated and inserted into a promotorless luciferase reporter vector, pGL3-Basic. Transfection experiments: Astrocytes were transfected cells using a modification of the method of Franzoso et al[ 16 ]. Briefly, 1.5 μg of DNA containing either pGL3-MIP2 or pGL3-basic were incubated in HBS solution (137 mM NaCl, 5 mM KCl, 0.88 mM Na2HPO4, 20 mM Hepes) containing 250 mM CaCl2 for 10 minutes at room temperature. The mixtures were added in 2 mL of media to astrocytes that were nearly confluent. After a 16-hour incubation in a humidified atmosphere at 37 C° containing 5% CO2, cells were washed to remove debris and cultured for an additional 24 hours. LPS plus or minus curcumin (10- 4 M to 10- 7 M) was then added and transfected cells were further cultured for 24 hours. At the conclusion of culture, cells were harvested, cell lysates were prepared, and lysates were analyzed using a luciferase assay system (Promega, Madison, WI) in accordance with the manufacturer's instructions. Results and discussion To determine whether mechanisms apart from its well-documented anti-oxidant activity might provide possible neuroprotection against inflammation-mediated injury, we investigated the effect of curcumin on astrocyte production of the chemokine MIP-2 in response to LPS. In initial experiments, we found that optimal MIP-2 production occurred when confluent astrocyte cultures were stimulated with 5 μg/ml of LPS during a 16-hour culture (data not shown). Culturing such astrocytes with a dose of curcumin (10- 5 M) that had no effect on viability as measured by LDH release (data not shown), abrogated LPS-stimulated MIP-2 production (Figure 2 ). Figure 2 LPS-induced MIP-2 production is inhibited by curcumin. Confluent astrocytes were cultured in medium alone or were stimulated with LPS (5 μg/ml) in the presence (LPS + Curcumin) or absence (LPS) of curcumin (10 -5 M). The supernatants were collected after 16 hours and MIP-2 protein (in picograms/ml) was measured by ELISA. Data are the mean ± standard deviation of 4 experiments. Mean MIP-2 production in the medium and LPS + curcumin groups differ significantly from that in the LPS group (p < 0.001 by Student's t test). Mean MIP-2 production does not differ significantly between the medium and LPS + curcumin groups (p > 0.2 by Student's t test). The effect of curcumin on LPS-induced production of MIP-2 mRNA was examined next. Preliminary experiments showed that optimal message for MIP-2 in response to LPS occurred after 4 hours of culture in astrocytes (data not shown). As was true for MIP-2 protein, culture of astrocytes with curcumin (10- 5 M) markedly inhibited chemokine gene expression in response to LPS (Figure 3 ). Figure 3 LPS-induced MIP-2 mRNA expression is inhibited by curcumin. Confluent astrocyte cultures were stimulated with LPS (5 μg/ml) in the presence or absence of varying doses of curcumin (10 -5 -10 -7 M) or vehicle (0.05% ethanol), mRNA was then extracted, reverse transcribed and amplified using a mouse MIP-2 primer. To determine whether curcumin inhibits MIP-2 gene transcription, a construct was created in which 537 base pairs of the MIP-2 promoter, spanning nucleotides -539 to -2 of the MIP-2 gene (see Methods), were fused to a promoter-less luciferase reporter gene (pGL3-MIP-2, Figure 1 ). As shown in the representative experiment in Figure 4 , curcumin abrogated LPS-stimulated MIP-2 gene expression in transiently transfected astrocytes. In three separate experiments, essentially complete inhibition of LPS-induced MIP-2 gene expression (100%, 92%, 94%) was observed with curcumin in doses of 2 μM. Figure 4 Curcumin inhibits the activity of MIP-2 at the level of gene transcription. Confluent astrocyte cultures were transfected with 1.5 μg of pGL3-MIP-2 or pGL3-Basic and stimulated with LPS (5 μg/ml) in the presence or absence of varying amounts of curcumin (10 -4 -10 -6 M). Transfected cells were harvested and luciferase activity in the cell lysates was quantified. The dose of curcumin is shown in log scale. Results are representative of three experiments. As a specificity control, the effect of EGCG, a catechin present in green tea with potent anti-oxidant activity, was examined on MIP-2 gene expression in astrocytes. In contrast to curcumin, EGCG in doses as high as 10- 3 M had no effect on LPS-stimulated MIP-2 mRNA expression (Figure 5 ). The results suggest that the inhibitory effect of curcumin on MIP-2 production may not be due to its anti-oxidant properties. Figure 5 Curcumin but not EGCG inhibits MIP-2 mRNA expression. Confluent cultures of astrocyte were stimulated with LPS (5 μg/ml) in the presence or absence of varying doses of curcumin (10 -5 -10 -7 M), EGCG (10–3), or appropriate vehicle (0.05% ethanol for curcumin, DMSO for EGCG). mRNA was then extracted, reverse transcribed and amplified using mouse MIP-2 primers. The study presented herein shows for the first time that curcumin is a potent inhibitor of inducible MIP-2 production by astrocytes, which are a major source of this chemokine in the brain[ 9 ]. In transient transfection experiments of astrocytes, virtually complete inhibition of MIP-2 inducible gene expression was observed with 2 μM curcumin. Since blood levels of curcumin approximating 2 μM were shown by Yang, et al[ 17 ] to block amyloid aggregation in a transgenic model of Alzheimer's disease, we believe that our data may have in vivo relevance. Transfection experiments in macrophages using a promoter, reporter-gene construct that contains canonical NFκB and NF-IL-6 cis-acting elements demonstrate that inhibition of MIP-2 by curcumin occurs at the level of gene transcription. The importance of either of these elements in the regulation of inducible MIP-2 gene expression in astrocytes remains to be determined. In some systems, inhibition of NFκB per se by curcumin is sufficient to abrogate gene expression. Thus, curcumin and its hydrogenated metabolites were shown to completely suppress transcription of nitric oxide synthase through down regulation of IκBkinase and NFκB activation in macrophages[ 18 ]. However, considering the fact that NFκB activation is linked to multiple upstream signaling pathways[ 19 ] and that curcumin has been shown to suppress a number of inflammatory signaling cascades[ 20 ], inhibition mediated by this spice principle may be quite complex and highly variable, depending on the cell type and the activating stimulus. Inhibition of chemokine production represents a novel, potential mechanism by which curcumin may confer neuroprotection in CNS disorders characterized or accompanied by leukocytic infiltration. As stated above, MIP-2 is a dominant, driving force in the pathogenesis of many CNS disorders that are associated with infiltration of neutrophils in the brain[ 9 , 10 ]. Experimentally, recruitment of neutrophils to the CNS is followed by a breeching of the blood-brain barrier that is especially severe after administration of MIP-2[ 21 ] and may further contribute to inflammation by causing indiscriminate entry of leukocytes into the brain. The possible contribution of inflammatory infiltrates to neuronal injury is best illustrated by experimental studies in which MIP-2 activity was neutralized. For example, administration of anti-MIP-2 antibody to rats infected with Hemophilus influenza type b abrogated the influx of neutrophils to the meninges, ventricular system, and the periventricular areas of the brain and substantially decreased neuronal damage[ 11 ]. In addition to astrocytes, microglial cells and endothelial cells may be potential sources of MIP-2 production in pathological states of the brain. Stimulation of brain microvascular endothelial cells with tumor necrosis factor alpha (TNFα), induces the release of MIP-2 within 4 to 8 hours of in vitro culture[ 22 ]. Since TNFα levels in the brain are significantly elevated in traumatic brain injury (TBI), it remains possible that cytokine-mediated release of MIP-2 by endothelial cells, particularly those which comprise the blood brain barrier, may predispose to intracerebral neutrophil accumulation and neuronal injury in TBI. Similarly, in a model of hypoxia/reoxygenation, large increases in MIP-2 mRNA and protein were demonstrated in microglial cells suggesting a possible mechanism to account for PMN accumulation and inflammation in cerebral ischemia. Apart from its ability to inhibit MIP-2 production, curcumin's pleotropic antiinflammatory and anti-oxidative properties suggest its possible use in diseases of the brain accompanied by inflammation. Thus, LPS stimulation transcriptionally upregulates inducible nitric oxide synthase and cyclooxygenase-2 genes in microglia. This leads to the synthesis of nitric oxide (NO) and prostaglandins (PGs), respectively, and the possible formation of neuron-damaging free radicals, such as peroxynitrite. Curcumin abrogates the production of both NO and PGs in LPS activated microglial cells[ 20 ]. In a recently completed Phase I clinical trial, oral curcumin at a daily dose of 3.6 grams was, in general, well-tolerated and decreased inducible PGE 2 production in blood samples taken 1 hour after dose on days 1 and 29 of treatment by approximately 60%[ 23 ]. Consistent with its possible use in neurodegenerative diseases associated with oxidative stress injury, curcumin has been reported to decrease oxidative damage and amyloid deposition in a transgenic mouse model of Alzheimer's disease[ 24 ], and to reverse Aβ-induced cognitive deficits and neuropathology in rats[ 25 ]. In summary, the capacity of curcumin to inhibit astrocyte production of MIP-2, together with its broad immunosuppressive activities, strongly support the potential use of this spice principle in the treatment of inflammatory diseases of the CNS. List of abbreviations EAE, experimental allergic encephalitis; EGCG, epigallocatechin gallate; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MIP-2, macrophage inflammatory protein-2; NFκB, nuclear factor kappa B; NO, nitric oxide; PG, prostaglandin; pGL3-MIP-2, a reporter gene construct containing the MIP-2 promoter; PMN, polymorphonuclear leukocyte; TBI, traumatic brain injury; TNFα, tumor necrosis factor alpha. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MT participated in experimental design, acquisition of data, supervised all experiments, and carried out isolation of astrocytes, ELISA and transfection assays. BH isolated and amplified the MIP-2 gene promoter, and generated the MIP-2 promoter construct, pGL3-MIP-2. CS participated in culture of astrocytes and PCR analysis of MIP-2 gene. TS conceived of the study, participated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553992.xml |
554757 | Aggressive HIV-1? | New York City health officials announced on February 11, 2005 that a patient rapidly developed full-blown AIDS shortly after being diagnosed with a rare, drug-resistant strain of HIV-1. The New York City Department of Health issued an alert to all hospitals and doctors and a press conference was held to announce the emergence of an aggressive HIV-1 strain that may be difficult to treat and that appears to trigger rapid progression to AIDS. Is the panic justified? | The two phenomena of rapid disease progression and multi-drug resistance, which are combined in the aggressive HIV-1 strain, are not unique. HIV-1 causes a persistent infection and this virus is generally not a fast killer. Within the Amsterdam Cohort Studies on HIV-AIDS, it takes on average 8.3 years from the time a person is first infected with HIV-1 for AIDS to develop, and another 17 months from AIDS to death [ 1 ]. However, the length of the incubation period varies from 2 months to more than 20 years. Cases where it takes much shorter are not uncommon (rapid-progressors), and likewise there is a significant group of socalled long-term non-progressors [ 2 - 5 ]. There seems uncertainty about the actual date of infection of the New York individual, such that AIDS may actually not have developed within 2 to 3 months, but rather within 20 months, which makes this case less exotic. Transmission of a drug-resistant HIV-1 variant is not uncommon either [ 6 , 7 ]. The number of cases have remained relatively small, but may be on the rise due to an increase in therapy failures [ 8 ]. The New York virus appeared resistant to three classes of antivirals (the Reverse Transcriptase inhibitors; nucleoside and non-nucleoside drugs, as well as Protease inhibitors), but this is not unexpected either in the era of combination-therapy in which therapy failure will usually mean the emergence of multi-drug resistant HIV-1 variants. Drug-resistant HIV-1 variants usually have reduced replication capacity compared to a wild-type virus due to the mutations in the Reverse Transcriptase and Protease enzymes [ 9 ]. This loss in replication fitness may be even larger for a multi-drug resistant virus [ 10 ]. How does this relate to the aggressive disease course? More research is needed to resolve this issue. First, it is not always true that the acquisition of drug-resistance mutations causes a fitness loss. Even in case a loss is apparent, the virus may select compensatory changes over time, and the end result may in fact be a virus variant with increased replication fitness [ 11 ]. Second, one can only link a particular pathogenicity phenotype to a virus strain when a distinct disease pattern is seen in multiple infected persons. When an isolated case is discussed, it is equally possible that the particular disease pattern is not due to the virus, but rather due to a special property of the infected human host. Person-to-person variation in the immune system or other factors that interact with HIV-1 (receptors, innate immune factors etc) can greatly influence disease progression [ 12 ]. Overall, this case seems relatively rare but not necessarily alarming. Increased attention is not necessarily bad, but press conferences should be reserved for situations when a cluster of such transmissions is apparent. The current hype about super aggressive HIV-1 strains seems unfounded. Competing interests The author(s) declare that they have no competing interests | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554757.xml |
552326 | A giant subcutaneous leiomyosarcoma arising in the inguinal region | Background Subcutaneous leiomyosarcoma is a rare condition that accounts for 1% to 2% of all superficial soft tissue malignancies. Approximately 10% of cases arise in the trunk, although the extremities are the most commonly affected. Case presentation We report herein the case of a 31-year-old man with a subcutaneous leiomyosarcoma, measuring 124 × 105 mm, arising in the left inguinal region. A wide local excision (with a resection margin ≥ 20 mm) was performed. Histological examination of the resected specimen revealed a leiomyosarcoma with high cellularity and two mitoses per 10 high-power fields. The patient remains well with no evidence of disease 5 years and 8 months after the operation. Conclusion This is the first reported case of subcutaneous leiomyosarcoma arising in the inguinal region and also one of the largest tumors reported. The experience of this case and a review of the English-language literature (PubMed, National Library of Medicine, Bethesda, MD, USA) suggest that a resection margin of ≥ 10 mm is recommended when excising this rare tumor. | Background Subcutaneous leiomyosarcoma arises from smooth muscle in the walls of arterioles and veins. It is a rare tumor accounting for 1% to 2% of all superficial soft tissue malignancies [ 1 - 4 ]. It usually occurs in patients between 50 and 70 years of age [ 1 , 2 , 5 ], with a male predominance ranging from 2:1 to 3:1 [ 2 , 4 ]. Although most tumors present as a subcutaneous nodule in the extremities, usually measuring 30 mm or less in diameter, about 10% of cases arise in the trunk [ 1 , 2 , 4 ]. We herein report the case of a patient with a giant subcutaneous leiomyosarcoma arising in the inguinal region. Case presentation A 31-year-old man presented with a painless left inguinal tumor, which had gradually grown during the past six months. Physical examination on admission revealed a fist-sized subcutaneous tumor in the inguinal region. The overlying skin appeared normal without ulceration (Figure 1 ), and there was no inguinal lymphadenopathy. Computed tomography depicted a solid tumor with heterogeneous contrast enhancement in the adipose tissue, and no metastases to the liver and lung (Figure 2 ). With a tentative diagnosis of soft tissue sarcoma of unknown origin, a wide local excision (with a resection margin ≥ 20 mm) was performed. Figure 1 A fist-sized tumor arising in the subcutaneous adipose tissue in the inguinal region. An arrow indicates the navel. Figure 2 Computed tomography depicted a solid tumor with heterogeneous contrast enhancement (arrowheads) in the adipose tissue. The resected tumor, measuring 124 × 105 mm, was solid, encapsulated, and a homogeneous yellowish white in color, without central necrosis and hemorrhage on its cut surface. Routine histological examination with hematoxylin-and-eosin revealed that the tumor comprised spindle-shaped cells with high cellularity in parts (Figure 3 ) and two mitoses per 10 high-power fields. Immunohistochemistry with mouse monoclonal antibodies against desmin (D33, Dako Cytomation Japan Co. Ltd., Kyoto, Japan), alpha-smooth muscle actin (1A4, Dako Cytomation Japan Co. Ltd., Kyoto, Japan), vimentin (V9, Dako Cytomation Japan Co. Ltd., Kyoto, Japan) and S-100 protein (2A10, IBL-Japan Co. Ltd., Takasaki, Japan) was performed. As the tumor cells showed only immunoreactivity for desmin (Figure 4 ) and alpha-smooth muscle actin, a diagnosis of leiomyosarcoma of subcutaneous adipose tissue origin was confirmed. Figure 3 The tumor comprised spindle-shaped cells with high cellularity in parts (hematoxylin-and-eosin; original magnification, × 100). Figure 4 Spindle-shaped cells showing strong immunoreactivity for desmin in the cytoplasm (Desmin immunohistochemistry; original magnification, × 400). The patient had an uneventful recovery and was discharged on the 9th postoperative day. As the resection margin was negative, no adjuvant treatment was given. He remains well with no evidence of disease 5 years and 8 months after excision. Discussion Although subcutaneous leiomyosarcoma commonly arises in the lower extremities, it occasionally affects the trunk [ 1 , 2 , 4 ]. A review of the English-language literature (PubMed, National Library of Medicine, Bethesda, MD, USA) suggests that the case reported here is the first one arising in the inguinal region and involves one of the largest tumors reported thus far [ 2 , 7 ]. Earlier authors proposed several prognostic factors for soft tissue sarcomas (including subcutaneous leiomyosarcoma) [ 2 , 6 - 8 ]. Factors adversely affecting the prognosis include high mitotic index (≥ 5 mitoses per 10 high-power fields) [ 2 ], high histologic grade [ 6 ], extensive necrosis [ 7 ], nodular growth pattern [ 7 ], deep tumor [ 8 ], and large tumor size [ 8 ]. Among them, tumor size (≥ 5 cm) is the strongest independent prognostic factor [ 8 ]. In the current case, low mitotic index, low histologic grade and the absence of necrosis favored a good prognosis, while large tumor size, deep tumor and nodular growth pattern were adverse prognostic factors. As subcutaneous leiomyosarcoma is resistant to radiotherapy and chemotherapy [ 2 , 3 , 9 ], surgical excision provides the only chance of cure. Prognosis after excision is generally considered poor, and even after a wide excision, local recurrence may occur in 40% to 60% of patients, followed by distant metastases in 20% to 40% of cases [ 1 , 2 ]. Contaminated margins contribute to the frequency of local recurrences [ 5 , 10 - 12 ]. In the current case, the resection margin of ≥ 20 mm successfully controlled the tumor. McKee et al ., [ 12 ] demonstrated that resection margins ≥ 10 mm decreased the risk of both local and distant recurrences in patients with soft tissue sarcomas (including subcutaneous leiomyosarcoma). A resection margin of ≥ 10 mm is therefore recommended when excising this rare tumor. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KY , YS , NF , and DS took part in the operation, performed the literature search and drafted the manuscript for submission. HU performed histological examination. KH supervised the preparation of the manuscript and edited the final version for publication. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552326.xml |
553979 | Medicinal herb use among asthmatic patients attending a specialty care facility in Trinidad | Background There is an increasing prevalence of asthma in the Caribbean and patients remain non-compliant to therapy despite the development of guidelines for management and prevention. Some patients may self-medicate with medicinal herbs for symptomatic relief, as there is a long tradition of use for a variety of ailments. The study assessed the prevalence of use and the factors affecting the decision to use herbs in asthmatic patients attending a public specialty care clinic in Trinidad. Methods A descriptive, cross-sectional study was conducted at the Chest Clinic in Trinidad using a de novo , pilot-tested, researcher-administered questionnaire between June and July 2003. Results Fifty-eight out of 191 patients (30.4%) reported using herbal remedies for symptomatic relief. Gender, age, ethnicity, and asthma severity did not influence the decision to use herbs; however, 62.5% of patients with tertiary level schooling used herbs, p = 0.025. Thirty-four of these 58 patients (58.6%) obtained herbs from their backyards or the supermarket; only 14 patients (24.1%) obtained herbs from an herbalist, herbal shop or pharmacy. Relatives and friends were the sole source of information for most patients (70.7%), and only 10.3% consulted an herbalist. Ginger, garlic, aloes, shandileer, wild onion, pepper and black sage were the most commonly used herbs. Conclusions Among patients attending the Chest Clinic in Trinidad the use of herbal remedies in asthma is relatively common on the advice of relatives and friends. It is therefore becoming imperative for healthcare providers to become more knowledgeable on this modality and to keep abreast with the latest developments. | Background Recent reports from the Caribbean suggest that the incidence of asthma is following the global trend of increasing prevalence. In Jamaica, a prevalence of 20.8% for exercise-induced asthma was estimated in a cross-sectional study in schoolchildren [ 1 ]. About one in ten patients attending an Accident and Emergency Department in Trinidad were treated for acute severe asthma [ 2 ] and over 15,000 patients attended four A&E departments throughout the island over a 12-month period [ 3 ]. Inhaled corticosteroids as prophylaxis and 'as required' bronchodilator for symptomatic relief are established modalities for asthma management and prevention and the Commonwealth Caribbean Medical Research Council/Global Initiative for Asthma guidelines were adopted in the Caribbean in 1997 [ 4 ]. It has been noted that inefficient management predisposes patients to frequent hospitalization and reduced quality of life. In Trinidad, non-compliance and inadequate inhaler technique negatively impact on effective disease management [ 5 , 6 ]. The frequent unavailability of medication at public health facilities and the prohibitive cost at private pharmacies are significantly associated with non-compliance and consequently poor disease control. In these studies, some patients indicated their use of herbal remedies as an alternative to conventional medicines. Over the last few decades, a global resurgence in the use of herbal remedies has fuelled the growing multi-billion dollar international trade of botanical products. Many patients, dissatisfied with conventional medicines because they expect permanent cures, believe that herbal remedies are 'natural' and sometimes self-medicate without informing their attending physician. Although there is a long history of traditional use of medicinal herbs throughout the Caribbean [ 7 , 8 ] few studies were done to assess the prevalence of use. Surveys in Jamaica reported an almost 100% use of herbal teas and remedies by respondents throughout the island [ 9 ] and 71% in paediatrics inpatients at the University Hospital [ 10 ]. These studies, however, assessed only the lifetime use of medicinal herbs and did not identify their use for any particular disease. In Trinidad and Tobago, the use of 'bush medicine' in diabetic patients attending primary healthcare facilities throughout the island was assessed and although 42% reportedly used herbs, only 24% used this healthcare modality for self-management of diabetes [ 11 ]. Another survey conducted at an outpatient surgical facility in Trinidad indicated a lifetime prevalence of 86% among patients [ 12 ] for any healthcare issue. This study was undertaken to assess the extent of use of herbal remedies by asthmatic patients attending a specialty chest clinic in Trinidad for symptomatic relief and to determine the factors influencing the patient's decision to use herbs. Methods The study was approved by the Ethics Committee of the Faculty of Medical Sciences, University of the West Indies, St. Augustine campus and permission to interview patients was granted by the Director of the Chest Clinic of the Ministry of Health, Trinidad and Tobago. The study was conducted over the two-month period June to July 2003. Sample and setting The Chest Clinic was chosen as the source of subjects as this is the only national tertiary level health facility specializing in the management of respiratory diseases. Patients entering the study were physician-diagnosed asthmatics based on self-reporting symptoms of wheezing, chest tightness and nocturnal coughing in the previous year. Patients were recruited by consecutive sampling and the nature and purpose of the study were explained on an individual basis. Those confirming their willingness to participate signed their informed consent and were interviewed using a de novo , pilot-tested, researcher-administered questionnaire. Interview instrument The questionnaire assessed demographic data such as age, gender, ethnicity, residential district, education, employment and socioeconomic status. Subjects reported their disease severity as intermittent, moderate or severe as determined by the Global Initiative for Asthma (GINA) guidelines with respect to symptom frequency [ 4 ]. Patients also reported their use of herbal remedies, identified the herbs used, the frequency of use, source of herbal medicines and the reasons for the use of herbs. Statistical analysis The sample size was calculated as 185 patients assuming a prevalence of 86% [ 13 ] with a confidence level of 95%. Since all variables were categorical, χ 2 tests were performed to determine whether there were statistically significant associations between the use of herbs and these variables. The p value was set at <0.05 for statistical significance. The data was analyzed using SPSS for Windows (Version 9.0, Chicago, IL). Results Demography During the study period one hundred and ninety one patients consented to participate. The demographic details of the sample are given in Table 1 . Patients between 35 and 64 years of age formed the largest portion of the sample (62.3%). There was a significant gender difference with females outnumbering males by a 2:1 ratio, p < 0.01. Most patients were of Asian Indian origin (58.1%) and resided in suburban areas (60.2%). There was a high level of unemployment (30.4%); this could be correlated to primary schooling (seven or less years of formal education) being the highest educational level attained in 52.9% and no formal schooling in 5.2% of the sample population. Income was low, with 42.9% of the sample population earning below US$4,000 per year. Table 1 Demographic details of patient sample Factors (n = 191) No. (% of sample) No. (%) using herbs Gender Male 61 (31.9) 19/61(31.2) Female 130 (68.1)* 39/130 (30.0) Age groups 16–34 34 (17.8) 11/34 (32.4) 35–50 52 (27.2) 21/52 (40.4) 51–64 67 (35.1) 18/67 (21.1) ≥ 65 38 (19.9) 8/38 (13.8) Ethnicity African 45 (23.6) 13/45 (28.9) Asian Indian 110 (57.6)* 31/110 (28.2) Mixed 35 (18.3) 14/35 (40.0) Other 1 (0.5) 0/1 (0.0) Asthma severity Intermittent 90 (47.1) 29/90 (32.2) Mild Persistent 29 (15.2) 9/29 (31.0) Moderate Persistent 27 (14.1) 7/27 (25.9) Severe Persistent 45 (23.6) 13/45 (28.9) Antiasthmatic drug use The GINA guidelines were recently adopted in the Caribbean and asthmatic patients are currently treated according to their symptom severity. In our sample population, particularly in patients with moderate and severe symptoms, corticosteroids (controllers) and β 2 -agonists (relievers) were prescribed at very high rates, Table 2 . Almost 90% of all patients with moderate symptoms were prescribed drugs in these classes. Almost all patients with severe symptoms were prescribed β 2 -agonists. This high level of prescription and use of β 2 -agonists suggest a lack of symptomatic control in our sample population. Theophylline and anticholinergics were prescribed in both categories of patients, but to a lesser extent. Table 2 Antiasthmatic drug use and self-reported compliance in patient sample Drug Use & Compliance Asthma Severity Moderate Persistent (n = 27) Severe Persistent (n = 45) Corticosteroids 24 (88.9) 39 (86.7) β 2 agonists 24 (88.9) 44 (97.8) Anticholinergics 5 (18.5) 13 (28.9) Theophylline 9 (33.3) 19 (42.2) Self-reported Compliance 27 (100) 43 (95.6) Factors influencing the use of herbal remedies Gender, age, ethnicity, residential district, employment status, income and asthma severity had no statistically significant effect on the use of herbal remedies within the sample population, Table 3 . However, almost two-thirds (62.5%) of patients with tertiary education used herbal remedies for asthma, p = 0.025. Table 3 Socioeconomic details of patient sample Factors (n = 191) No. (% of sample) No. (%) using herbs Residential district Rural 27 (14.1) 6/27 (22.2) Suburban 119 (62.3)* 36/119 (30.3) Urban 42 (22.0) 15/42 (35.7) No response 3 (1.6) 1/3 (33.3) Highest educational level attained No formal education 10 (5.2) 2/10 (20.0) ≤ 7 years of formal education 101 (52.9) 26/101 (25.7) ≤ 12 years of formal education 64 (33.5) 20/64 (31.3) > 12 year of formal education 16 (8.4) 10/16 (62.5)* Employment status Unemployed 58 (30.4) 19/58 (32.8) Technical 12 (6.3) 6/12 (50.0) Professional 16 (8.4) 5/16 (31.3) Clerical 13 (6.3) 6/13 (46.2) Vocational 28 (12.6) 5/28 (17.9) Student 8 (4.2) 4/8 (50.0) Housewife 23 (12.0) 7/23 (30.4) Pensioner 33 (17.3) 6/33 (18.2) Annual income (US$) ≤ 3,999 82 (42.9) 22/82 (26.8) 4000 – 9,999 73 (38.2) 23/73 (31.5) 10,000 – 11,999 12 (6.3) 4/12 (33.3) 12,000 – 19,999 15 (7.9) 7/15 (46.7) ≥ 20,000 9 (4.7) 2/9 (22.2) Characteristics of patients using herbal remedies Most patients (70.7%) using herbs were advised by a relative or friend and only 10.3% sought the advice of an herbalist, Table 4 . A cultural/traditional basis was the reason for herbal remedy usage in twenty-one (36.2%) patients and another twelve (20.7%) patients used herbs because they felt that were either 'natural' or 'healthy'. Twelve (20.7%) patients used herbs because they believed that their physician-prescribed allopathic medicines were not working. Table 4 Characteristics of patients using medicinal herbs (n = 58) Patient characteristics No. (%) (Out of 58) Reason for using herbs Traditional/Cultural 21 (36.2) Natural/Healthy 12 (20.7) Conventional medicine not working 12 (20.7) Other 13 (22.4) Source of herbs At home/backyard garden 25 (43.1)* Supermarket 9 (15.5) Herbalist/Herbal Shop/ Pharmacy 14 (24.1) Relative/friend 4 (6.9) Other 6 (10.3) Source of information Relative/friend 41 (70.7)* Herbalist 6 (10.3) No consultation 9 (15.5) Other 2 (3.5) Time last used herbs Within last week 17 (29.3) Within last month 9 (15.5) Within last 3 months 6 (10.3) Within last 6 months 3 (5.2) More than 6 months ago 23 (39.7) Most patients (58.6%) obtained their herbs or medicinal plants from either their backyards or the supermarket. Only fourteen (24.1%) obtained their herbal supplies from an herbalist, herbal shop or pharmacy. Seventeen (29.3%) of these patients reported using herbs within the last week and most these patients (60.3%) used herbs within the last six months. Many of these patients were using both physician-prescribed antiasthmatic drugs and herbal remedies, Table 5 . No patient with either moderate or severe symptoms indicated that herbal remedies alone were sufficient to relieve symptomatic episodes. It is interesting to note that most patients with moderate symptoms (57.1%) believed that concurrent use of conventional medications and herbs gave better symptomatic relieve. One the other hand, most patients with severe symptoms (53.8%) believed that physician-prescribed medications worked better than herbal remedies, while 23.1% believed that neither relieved their symptoms. Table 5 Antiasthmatic drug use and self-reported compliance in patients using herbal remedies Drug Use & Compliance Asthma Severity Moderate Persistent (n = 7) Severe Persistent (n = 13) Corticosteroids 6 (85.7) 10 (76.9) β 2 agonists 6 (85.7) 13 (100) Anticholinergics 1 (14.3) 5 (38.5) Theophylline 2 (28.6) 7 (53.8) Self-reported Compliance 5 (71.4) 8 (61.5) Subjective Benefits of therapy Herbal remedies alone better 0 (0) 0 (0) Herbal remedies and drugs better 4 (57.1) 3 (23.1) Drugs alone better 3 (42.9) 7 (53.8) Neither herbs and/or drugs work 0 (0) 3 (23.1) Herbs used in asthma Most patients in the sample used more than one medicinal herb simultaneously, which were usually prepared and administered as mixtures in teas. Almost one in four patients using medicinal herbs (22.5%) used either garlic ( Allium sativum ) or ginger ( Zingiber officinale ) for symptomatic relief of asthma, Table 6 . Aloes ( Aloe vera ) shandileer ( Leonotis nepetifolia ), wild onion ( Hymenocallis tubiflora ), pepper ( Capsicum spp .) tulsi ( Ocimum gratissimum ), black sage ( Cordia curassavica ), shadon beni ( Eryngium foetidium ), lemongrass ( Cymbopogon citratus ) and nutmeg ( Myristica fragrans ) were the more popular traditional indigenous West Indian medicinal plants used. Two patients reported using marijuana (leaves and roots). Herbs of European and North American origin, identified as Echinacea ( Echinacea purpurea ), Golden Seal ( Hydrastis canadensis ) and Chamomile ( Matricaria chamomilla ) were less frequently used. Five patients reported using trade name imported tablets for asthma. Table 6 Medicinal plants commonly used by respondents (n = 58) using herbal remedies, ranked by prevalence Common name Botanical name n % Garlic Allium sativum L. 13 22.4 Aloes Aloe vera (Aloe barbadensis Miller) 13 22.4 Ginger Zingiber officinale Roscoe 9 15.5 Shandileer Leonotis nepetifolia (L.) R.Br. 8 13.8 Wild Onion Hymenocalis tubiflora 8 13.8 Pepper Capsium spp . L. 6 10.4 Black Sage Cordidia cylindristachya R.S. 6 10.4 Tulsi Ocimum grastissimum L. 5 8.6 Echinacea Echinacea purpurea L. Moench 5 8.6 Shadon beni Eryngium foetidium L. 5 8.6 Nutmeg Myristica fragrans Houtt. 4 6.9 Lemongrass Cymbopogon citratus (DC.) Stapfl 4 6.9 Christmas bush Chromolaena odorata (L.) R.M. King & H. Rob. 4 6.9 Golden Seal Hydrastis canadenesis L. 3 5.2 Bayleaf Pimenta racemosa (P. Mill) J.W. Moore 3 5.2 Charmomile Matricaria chamomilla L. 3 5.2 Hibiscus Hibiscus rosa-sinensis Linn. 3 5.2 Noni Morinda citrifolia Linn. 3 5.2 Marijauna Cannabis sativum L. 2 3.5 Effect of income and education on the use of herbs Patients using easily accessible herbs such as ginger ( Zingiber officinale ) and aloes ( Aloe vera ), and traditional indigenous medicinal herbs such as shandileer ( Leonotis nepetifolia ) and tulsi ( Ocimum gratissimum ) were more likely to be earning less than US$12,000, Table 7 . Herbs of European or North American origin ( Echinacea purpurea and Matricaria chamomilla ) were more likely to be used by patients earning in excess of US$12,000 per annum. Income did not affect the use of either garlic or cocoa onion. Table 7 Income and education effects on use of herbs Medicinal herb used Percentage of patients with annual income Percentage of patients with formal education ≤ US$12,000 > US$12,000 ≤ 12 years > 12 years Ginger ( Zingiber officinale ) 18.4* 0.0 14.0 20.0 Garlic ( Allium sativum ) 22.5 22.2 16.7 50.0* Aloes ( Aloe vera ) 24.5* 11.1 25.0 10.0 Shandileer ( Leonotis nepetifora ) 16.3* 0.0 14.6 10.0 Cocoa Onion ( Hymenocalis tubiflora ) 10.2 11.1 10.4 10.0 Tulsi ( Ocimum grastissimum ) 10.2* 0.0 10.4 0.0 Golden Seal ( Hydrastis canadenesis ) 6.1 0.0 6.3 0.0 Echinacea ( Echinacea purpurea ) 4.1 33.3* 4.2 30.0* Chamomile ( Matricaria chamomilla ) 2.0 11.1* 3.9 10.0 Aloes ( Aloe vera ), tulsi ( Ocimum gratissimum ) and golden seal were preferred in patients with at least twelve years of formal education, Table 7 . Garlic and Echinacea were the preferred herbal medicines in patients with more than twelve years formal education. Educational level did not affect the patients' decision to use shandileer ( Leonotis nepetifolia ), wild onion ( Hymenocallis tubiflora ) or ginger ( Zingibe officinale ). Discussion This is the first study of its kind in the Caribbean to assess the use of medicinal herbs by asthmatic patients attending a specialty care clinic. The findings of this study are instructive as the use of medicinal herbs for self-medication in disease management has far reaching implications on the quality of healthcare delivery [ 14 ]. We report a prevalence of 30.4% in our patient sample, which is significantly higher than that in the UK, Denmark, Singapore and in the US [ 15 - 18 ]. Most patients using medicinal herbs relied on the advice of relatives and friends as their sole source of information, as were caregivers of children in a US study [ 19 ]. We suggest that this information on the use of medicinal plants could have come from traditional/cultural knowledge, anecdotal evidence or from the greater public awareness through information networks such as the internet on the potential medicinal benefits of herbs. Asthma is an emerging chronic disease in the Caribbean and we suggest that the traditional knowledge in this area may be relatively 'new' and exist in relation to other diseases affecting the respiratory tract, such as cough, the common cold and the flu. This may be one of the reasons for the low prevalence of use of herbs in elderly asthmatic patients, as a strong traditional knowledge may not have existed. We expected a higher prevalence of herbal use in individuals living in rural areas as these districts are depots for traditional knowledge as was reported in Jamaica where rural respondents used a larger variety of herbs than those living in urban areas [ 10 ]. As suggested earlier, we suspect that due to the recent emergence of asthma as a chronic disease in the Caribbean it is reasonable to expect that traditional knowledge in the management of this disease is not strong and our results are indicative of this. We suspected that employment status could have predicted the use of herbs, however, this was not the case in our study sample. Unemployed patients did not improvise more in their use of herbal remedies than those in other income groups, even though most of the herbs used were relatively common, readily available and cheap. The low socioeconomic status of the majority of the sample may have prohibited both consultation with qualified herbalists and the purchase of imported, processed herbs that would have incurred additional out-of-pocket expense to the patient. What we noted was that there was no difference in the use of herbs across the income ranges and that in fact, patients earning relatively modest annual incomes between $US12,000 and $US19,999 were most likely to use herbs, although this did not reach statistical significance. Attaining a higher education positively influence the decision to use herbs. We suggest that in the absence of traditional knowledge regarding the medicinal use of herbs for asthma, a higher educational level may predispose an individual to greater access to general knowledge, especially with greater exposure to the internet and other sources of information, and this could be a factor in positively influencing the individual's decision to use medicinal herbs. The availability of scientific evidence-based information on the efficacy of herbs for diverse healthcare problems may be particularly significant in patients with the resources to avail themselves to such information, particularly those with higher educational and income levels. This is particularly true for garlic and Echinacea, which have been extensively researched and furthermore patients with higher educational and income levels would be more likely be at an advantage to access information via literature or on the world wide web regarding the use of these medicinal plants. Patients using imported, processed, and obviously more expensive herbal medications were on the higher end of the socioeconomic scale and were more likely to afford these medications. It was also observed that garlic and Echinacea were the herbs of choice in patients with higher educational levels. These herbs have a long tradition of use and are widely researched in Europe and North America. The traditional use and strong scientific evidence to support their therapeutic efficacy could be important factors influencing the patient's decision. It has been suggested elsewhere that patients with higher educational levels also tend be more involved in the management of their health; they tend to self-medicate or even suggest to their physicians the course of therapy. Although one in five patients using medicinal herbs stated that "conventional medicines were not working" as the reason for using this alternative healthcare modality, we noted that asthma severity does not affect the decision to use herbs. In previous studies, poor management was associated with non-compliance with prescribed pharmacotherapy and poor inhaler technique [ 5 , 6 ]. The backyard and home garden were major sources of readily available herbs such as aloes, shadon beni and lemongrass. Wild growing 'weeds' such as shandileer, tulsi, cocoa onion and black sage were also identified. The supermarket was a major source of inexpensive common medicinal herbs such as garlic, ginger and nutmeg. The identification of these medicinal herbs provides an opportunity to investigate West Indian plants used to treat asthma to determine whether they possess pharmacological properties. Scientific investigations have shown that some of these herbs possess pharmacological and anti-inflammatory properties, and these may be useful in suppressing the characteristic exaggerated immune response in asthma [ 20 - 24 ]. Pepper and bayleaf have also been shown to exhibit anti-inflammatory properties [[ 25 , 26 ]27]. There is an imperative to commence scientific investigations on traditional West Indian medicinal plants to determine their therapeutic efficacy and safety. The survey instrument specifically asked questions on the use of medicinal herbs in asthma and did not inquire about the use of herbs as customary teas or tonics. We therefore did not determine lifetime prevalence for the use of herbs in our patient sample, but we suppose that had this been included that there might have been a prevalence similar to those reported in the Jamaica [ 10 , 11 ] and Trinidad [ 13 ] surveys. The survey was also limited in that by electing to conduct the study at a public health facility we obviously had a bias towards patients at the lower rung of the socioeconomic ladder, with lower income and educational status. As a consequence, the results reflected patients from this demographic background. We may have expected a different outcome in asthmatic patients attending private institutions, where their characteristics would have been slightly different, as we noted that even in our sample the small number of persons with higher income and educational status tended to use more medicinal herbs for symptomatic relief. We did not assess whether patients informed their attending physician at the clinic about their use of herbs or determined whether the knowledge or attitudes of these physicians regarding the use of herbs influenced the patients' decision to use herbs. The study was also limited in that we did not ascertain the out-of-pocket expense for herbal remedies by patients, although most stated that herbal medicines (which we supposed were processed, imported products) were more expensive than conventional medicines. We assumed that an additional expense would have only been incurred by those patients purchasing processed, imported herbs obtained from a herbalist, herbal shop or pharmacy (24.1%) and who actually consulted a herbalist (10.3%). We also reasoned that since all the other herbs used were inexpensive and available from either the backyard garden or supermarket (58.6%) that the cost to patients selecting these remedies was minimal. Conclusions The findings of this study are important in that local medicinal plants in Trinidad have been identified in the self-management of asthma in a significant number of patients attending the specialty clinic. These identified herbs can now be targeted for scientific investigation to determine whether their pharmacological efficacy will assist in the development of viable healthcare alternatives in a developing country. These findings are also important for policymakers in the health sector who are given the mandate to regulate issues pertaining to the public's health. We are also becoming more aware of the potential for critical interplay between herbs and drugs when taken concomitantly to produce life-threatening interactions. Since herbs are here to stay and patients will continue to self-medicate with increasing frequency, it is imperative that healthcare providers become more knowledgeable on this modality and keep abreast with the latest developments in herbal therapy. Competing interests The author(s) declare that they have no competing interests. Authors' contributions YNC was the P.I. in this study. He was responsible for the study concept, development of methodology, coordinating the research activities, analyzing the data, and writing the manuscript. AFW was responsible for data input and analysis. DA was involved in methodological development, data collection, data input and analysis and presentation at regional conference. RC was involved in methodological development, data collection, data input and analysis. NW was involved in methodological development, data collection and input. RM was involved in methodological development, data collection and input. OS was involved in methodological development, data collection and input. DW was involved in methodological development, data collection and input. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553979.xml |
544544 | Sex Determination across Evolution: Connecting the Dots | Sexual differentiation appears to be an ancient, and potentially homologous, feature of animal biology, and yet the pathways that underlie the process exhibit bewildering variety | Evolutionary developmental biology is motivated by the premise that the differences we see between species are caused by changes that have occurred in the genes that regulate their developmental programs. Beginning in the 1980s, general principles began to emerge about the evolution of development in animals. The identification of the Hox genes in Drosophila melanogaster and the subsequent discovery of their conservation and similar expression in different Metazoans led to the revolutionary realization that many of the mechanisms critical to basic animal development have been conserved across more than 500 million years of evolution. Many other developmental pathways, such as those specifying the heart and the central nervous system, have since been elucidated and promptly subjected to successful comparative analysis. These celebrated discoveries illustrate ways that very different organisms are, at a fundamental level, similar to one another. But not all developmental processes are so conservative; an outstanding example is sex determination. The majority of animal species produce two sexes, and current phylogenies (e.g., [ 1 ]) suggest that sexual dimorphism was likely a feature of the last common ancestor of the coelomate bilaterians, a vast clade of animals that excludes only sponges, ctenophores, cnidarians, and acoel flatworms. However, though critical for development and reproduction, the mechanisms that specify sex determination are among the least-conserved known. Marked variation exists in both the primary sex determination signal and in the downstream genetic pathways that interpret the signal. We are thus presented with our first conundrum: sexual differentiation appears to be an ancient, and potentially homologous, feature of animal biology, yet its genetic specification suggests multiple origins. Bewildering Variety The variety of primary sex determination cues was appreciated long before the advent of molecular genetics [ 2 ]. The two broadest categories are genetic sex determination (GSD), in which the sex of offspring is set by a sex chromosome or an autosomal gene, and environmental sex determination (ESD), in which sex is determined by temperature (as with turtles), local sex ratio (as with some tropical fish), or population density (as with mermithid nematodes). Though little is known about the molecular mechanisms of ESD, within the GSD systems many different mechanisms have been uncovered. Dual sex chromosome systems, in which either the female (ZW/ZZ) or the male (XX/XY) is heterogametic, are common, as are systems set by the ratio of the number of X chromosomes to sets of autosomes (X:A). There are also systems in which heterozygosity at a single locus is required for female development (known as complementary sex determination; [ 3 ]), as well as systems involving sex determination via multiple genes with additive effects. Molecular genetic investigations of GSD in model systems such as Drosophila , Caenorhabditis , and mice have revealed a clear lack of conservation, underscoring the diversity. For example, although the primary sex determination signal in both D. melanogaster and C. elegans is the X:A ratio, the fruit fly pathway consists of a cell-autonomous cascade of regulated mRNA splicing, while that of the nematode follows a Hedgehog -like intercellular signaling pathway [ 4 ]. GSD in mammals depends (with some interesting exceptions—see [ 5 ]) upon a Y-specific dominant gene ( Sry ) encoding a transcription factor. In the face of such impressive differences, perhaps we should question our assumption of homology: could it be that sex determination in different taxa has arisen independently over and over again in evolution? Until 1998, this seemed like a good bet. The discovery of the homology of the key sex-determining genes doublesex in Drosophila and mab-3 in C. elegans provided the first evidence for a common evolutionary basis of sex determination in animals [ 6 ]. Soon, related doublesex-mab-3 (DM)-family genes with roles in male sexual development were discovered in vertebrates and even cnidarians [ 7 , 8 ]. Here at last was a smoking gun that could link the diverse metazoan sex determination systems ( Figure 1 ). But as satisfying as the result was, it immediately gave birth to another mystery: if the enormous diversity of sex determination systems are all derived from a common ancestor, how could they possibly have been modified so radically? After all, sexual differentiation and reproduction are hardly unimportant developmental processes! Figure 1 Diverse Genetic Factors Converge on a Conserved Regulator The primary sex determination mechanisms are shown, from left to right, for Drosophila , Caenorhabditis , the box turtle Terrapene carolina , and humans. These proximate signals are then relayed by diverse signal transduction pathways that ultimately converge on a DM-family gene. The left image is from Muller [ 23 ]; the center-left image appears courtesy of Dr. Barbara Conradt, the center-right image appears courtesy of J.D. Willson, and the right image is from a plaque mounted on the NASA spacecraft Pioneer 11. Focusing on Close Relatives To understand how such diversity came to be, we need to look at the differences between closely related species. This approach allows the discovery and interpretation of small-scale sex determination changes before they are obscured by subsequent changes. The processes discovered in this way might then be reasonably extrapolated to explain the seemingly unrelated systems of more deeply diverged taxa. Work in dipterans [ 9 ] and nematodes [ 10 ] has revealed three evolutionary phenomena that characterize shorter-term sex determination evolution. The first of these is the often astounding rate of molecular evolution at the level of nucleotide and aminoacid sequences. Although some sex-determining genes are well conserved, many show unprecedented substitution rates [ 11 ]. An extreme example is the central integrator of the X:A ratio in Caenorhabditis , xol-1 . The xol-1 orthologues of the closely related nematodes C. elegans and C. briggsae are a mere 22% identical [ 12 ], even though genes surrounding xol-1 are much better conserved ( Figure 2A ). Remarkably, the 3′ neighbor of xol-1 , the immunoglobulin dim-1 , is only 5 kb away and is essentially identical between species. Figure 2 Evolutionary Dynamics of Sex-Determination Pathways (A) Rapid sequence evolution. Shown are the genes in xol-1 region of C. elegans that have syntenic homologues in C. briggsae , with the amino-acid-level identity between them indicated below. (B) Pathway evolution and primary signal swapping (modified from Graham et al. [ 9 ]). In Drosophila (L), the X:A ratio indirectly regulates tra splicing through a requirement for Sxl . In the medfly Ceratitis (R), Sxl is not a sex determination gene, and the female-promoting positive regulation of tra is instead autonomous. Its inhibition by the dominant M gene allows an XX/XY system to replace one based on the X:A ratio. (C) Convergent evolution of nematode hermaphroditism in C. elegans and C. briggsae . fog-2 exists only in C. elegans , and although all species use the fem genes for male somatic development, only C. elegans requires them for hermaphrodite spermatogenesis. A second phenomenon, best exemplified by dipteran insects, is the modification of genetic control pathways through the gain or loss of key pathway components ( Figure 2B ). In Drosophila , the first gene to respond to the X:A ratio is Sxl , whose transcription is regulated by both autosomal and X-linked factors very early in development [ 4 , 13 ]. When X: A = 1 (i.e., in female embryos), Sxl transcription occurs and produces Sxl protein. Later in development, transcription from a second promoter occurs in both sexes, but these transcripts cannot be productively spliced without the earlier burst of Sxl expression. As a result, only females sustain Sxl expression, and in turn only females can productively splice the mRNA of tra , its downstream target. Productive splicing of tra is required to produce the female-specific form of dsx , a founding member of the DM family mentioned above. In a series of groundbreaking papers, Saccone and colleagues investigated the pathway in the more distantly related heterogametic Mediterranean fruit fly Ceratitis capitata . The first surprise was that although a highly conserved Sxl homologue exists in Ceratitis , it does not undergo sex-specific regulation similar to that of Drosophila , which suggests that it does not play a key switch role (Saccone et al. 1998). Similar results have also been found for the housefly, Musca domestica [ 14 ], indicating that the role of Sxl in sex determination may be restricted to Drosophila and its closest relatives. In contrast, tra and dsx are key sex regulators in all dipterans examined thus far. A further surprise came when the Ceratitis tra homologue was characterized [ 15 ]. In the case of this gene, clear evidence for sex-specific regulation was found, and as with Drosophila , only females productively splice tra mRNA. However, this splicing difference can be explained nicely by a positive feedback, similar to that seen in Drosophila Sxl , in which Tra protein regulates its own splicing. In 2002, Pane et al. proposed that the dominant, male-specifying M factor on the Y chromosome inhibits this autoregulation [ 15 ]. As a result, males cannot make functional Tra protein, and the male form of Dsx is produced. These experiments show not only how a pathway can evolve, but also, importantly, how X:A and heterogametic GSD systems can be interconverted by modifying the cue that regulates a conserved molecular switch gene (the splicing of tra mRNA). A detailed scenario for how this might occur has recently been proposed [ 16 ]. Finally, recent studies of Caenorhabditis nematodes have shed light on the genetic basis of the convergent evolution of sex determination related to mating system adaptations. An important factor in this area are new phylogenies of the genus [ 17 , 18 ], which consistently suggest the surprising possibility that the closely related hermaphroditic species C. elegans and C. briggsae acquired self-fertilization independently, from distinct gonochoristic (male/female) ancestors ( Figure 2C ). Although this scenario is somewhat uncertain purely on parsimony grounds, recent work on the genetic control of the germline bisexuality that defines hermaphroditism has tipped the balance toward parallel evolution. Working with C. elegans , Clifford et al. [ 19 ] cloned fog-2 , a gene required for spermatogenesis in hermaphrodites but not in males. Upon doing so, it became clear that fog-2 is part of a large family of F-box genes and was produced by several recent rounds of gene duplication. The C. briggsae genome sequence suggested that while C. briggsae possesses a similarly large family of F-box proteins, the duplication event giving rise to fog-2 was specific to the C. elegans lineage. In this issue of PLoS Biology , Nayak et al. [ 20 ] extend this work by rigorously demonstrating that fog-2 is indeed absent in C. briggsae . The authors also identify a short, C-terminal domain that makes FOG-2 uniquely able to perform its germline sex-determining function. This domain is probably derived from a frame-shifting mutation in an ancestral gene. Working with C. briggsae , Stothard et al. [ 21 ], Haag et al. [ 22 ], and Hill et al. (unpublished data) have also found evidence of important species-specific regulation of germline sex determination. RNA interference and gene knockout approaches have shown that while C. elegans requires the male-promoting genes fem-2 and fem-3 to produce sperm in hermaphrodites, C. briggsae requires neither. Given that both genes have conserved roles in male somatic sex determination, this suggests that C. briggsae evolved hermaphroditism in a way that bypasses these genes. The long-standing mystery of sex determination and its diversity began by comparisons between distantly related species. Recent work on closer relatives has uncovered processes that through a reasonable extrapolation enable the connection of these disparate dots into a fascinating picture of developmental evolution. Though the divergence is extreme, it is likely that a better understanding of the evolution of sex determination genes and pathways holds lessons about the evolution of development in general. The next major challenge will be to integrate the comparative developmental data with the ecological and population processes that are driving the evolution of sex determination. Only then will we be able to say that the picture is complete. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544544.xml |
553986 | Nutritional knowledge, food habits and health attitude of Chinese university students –a cross sectional study– | Background We have previously shown that irregular lifestyle of young Japanese female students are significantly related to their desire to be thinner. In the present study, we examined the nutritional knowledge and food habits of Chinese university students and compared them with those of other Asian populations. Methods A self-reported questionnaire was administered to 540 students, ranging in age from 19-24 years. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. The parametric variables were analyzed using the Student's t -test. Chi-square analyses were conducted for non-parametric variables Results Our results showed that 80.5% of students had a normal BMI and 16.6 % of students were underweight with the prevalence of BMI>30 obesity being very low in this study sample. Young Chinese female students had a greater desire to be thinner (62.0%) than males (47.4%). Habits involving regular eating patterns and vegetable intake were reported and represent practices that ought to be encouraged. Conclusions The university and college arenas represent the final opportunity for the health and nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition. | Background The increasing problem of obesity has been observed in many lower-income countries during the last decades. China has adopted an open-market policy and experienced explosive economic growth, which has led to less food scarcity at the national level and to a remarkable transition in the structure of the diet of Chinese [ 1 ]. The composition of the Chinese diet has been shifting towards a diet higher in fat and meat, and lower in carbohydrates and fiber [ 2 ]. Additionally, decreased levels of physical activity and leisure are linked to increases in the prevalence of an overweight condition, obesity and diet-related non-communicable diseases [ 3 ]. In previous reports, we examined eating habits and dietary knowledge of female students in Japan. Our results showed that irregular lifestyle was significantly related to indefinite complaint, with the majority of students having a desire to be thinner although the prevalence of students who were overweight was very low in this study sample [ 4 ]. Universities and colleges are potentially important targets for the promotion of healthy lifestyles of the adult population. However, little is known concerning the body mass index (BMI) distribution and nutritional and health-related behavior of Chinese university students. The purpose of this study was to obtain a preliminary understanding of the relative level of BMI distribution of Chinese university students and to determine the nutritional knowledge and body-shape perceptions. Material and Methods This study was carried out between February 2001 and April 2002. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. A sample of 540 students aged 19 – 24 years were administered a self-reported questionnaire. The questionnaire consisted of 21 questions regarding eating, drinking and smoking habits (19 questions), with 2 questions related to dieting (trying to lose weight). Self-reported height and weight were used to calculate BMI (kg/m 2 ). The questionnaire was designed by the authors and based on a national dietary survey held by the Health and Labor Ministry of Japan. Some of the authors also traveled to China to investigate the dietary life of Chinese to facilitate questionnaire design. The questionnaire was first written in Japanese and then translated to Chinese utilizing fluent bilingual linguistic services. The translated Chinese version was back-translated to insure the original meaning was not lost. Informed consent was obtained from all participants of this study according to the Declaration of Helsinki. The statistical software package SPSS 10.0 was used for the analysis of data [ 5 ]. In this study, parametric variables were analyzed using the Student's t -test. Chi-square analyses were conducted for non-parametric variables. All analyses were two-tailed, and a 'p' value less than 0.05 was considered statistically significant. Results Characteristics of the sample and BMI categories The response rate was 96% (512 / 540). The characteristics of the subjects are shown in Table 1 . A total of 212 men and 300 women, with a mean age of 20 ± 1.9 years, participated in this study. The average height was 165.8 ± 7.8 cm, while the average weight was 56.9 ± 9.2 kg. Mean BMI was 20.6 ± 2.2. To analyze the distribution of BMI and health-related behavior, BMI was categorized into 4 groups according to mean BMI of ± 1 standard deviation (SD) (Figure 1 ). The average BMI for male students was 21.4 ± 2.5 and was highest in the categories 18.9≤BMI<21.4 (37.7%) and 21.4≤BMI<23.9 (32.5%). The average BMI for female students was 20.0 ± 1.8, with the categories 18.2≤BMI<20.0 (37.5%) and 20.0≤BMI<21.8 (31.4%) displaying high values. According to WHO BMI classifications [ 6 ], 97.1% of students were classified into the underweight or normal weight categories. 2.5% (13/512) students were overweight (BMI>25) and 0.4% (2/512) of students were obese (BMI>30). BMI values of deviations from the average sample show the presence of few extreme values. Eating habit The life style practices were compared by gender (see additional file 1 ). The majority of students (83.6 %) reported taking meals regularly, with 79.0% eating meals 3 times per day; there were no gender differences. However, a significant gender difference was found in the response relating to breakfast intake, with 66.8% of males and 82.3% of females reporting eating breakfast regularly (p < 0.0006). The frequency of snacking rate was significantly higher in females (31.1%) than in males (11.5%; p < 0.0001). The present sample demonstrated high consumption of vegetable and fruits. A total of 47.9% of students reported the consumption of colored vegetables such as spinach and carrots, and 32.5% of subjects reported eating fruit daily. Female students tend to eat more fruit than males (p < 0.0001). In addition, female students tend to eat with friends and family more frequently than males (p < 0.01). Few subjects smoke or drink alcohol. When the students eat out, female students are more likely to consider the calorie content of the menu than males (data not shown). Although 85.6% of students are aware of the concept of nutritionally balanced food, only a small number of students (7%) apply this concept when selecting food from a menu. Moreover, only 51% of students showed a desire to learn about healthy diets. Body image and health consciousness When subjects were asked about their history of dieting, 22.7% of respondents reported that they had dieted (see additional file 2 ). The proportion of female students having a dieting experience (29.8%) was more than twice as great as that of male subjects (12.7%; p < 0.0006). In total, 56% of the students selected 'thin or slim is beautiful'. The percentage by gender was, 47.4% for male and 62.0% for female students. Female students have a significantly greater desire to be thinner than males (p < 0.001). More than half of the respondents reported a desire to adopt healthier dietary habits. Moreover, a question regarding the degree of consciousness pertaining to health and diet was asked; 45.2% of male students and 48.3% of female students wish to learn about health and diet. Among female subjects, BMI<18.2 strongly showed their consciousness of health and diet (p < 0.03). Discussion This study aimed to determine the health, nutritional knowledge and dietary behavior of university students in China. As a result, we recorded the distribution of BMI among Chinese students and found a low prevalence of obesity, a finding that is consistent with a study of Japanese female students (BMI≥25 overweight was 5.8%, BMI>30 of obesity was 0%) [ 4 ]. In the United States, 35% of the college students are reported to be overweight or obese (BMI≥25) [ 7 ]. According to the WHO definition of obesity, BMI>30 is the cut-off point [ 6 ]. The definition is based on research of Caucasian populations. Asian populations are reported to have a higher body fat (%) at a lower BMI compared to Caucasians [ 8 ]. The WHO expert consultation reported that BMI in Asian populations is related to disease at a lower level [ 9 ]. In order to compare obesity prevalence between ethnic groups, BMI cut-off points for Asians need to be considered by well constructed and standardized body composition studies. It is notable that in China, the prevalence of overweight individuals increased from 1991 to 1997, with the increasing rate changing from 6.4 to 7.7 [ 10 ]. The proportion of energy derived from the fat of both vegetable and animal sources increased each year. A recent study revealed that energy derived from dietary fat accounted for more than 30% of the total energy [ 11 ]. Changes in dietary composition, which correspond to socioeconomic growth, may accelerate the prevalence of obesity in China. The results of our study show that the majority of students regularly eat three times per day, and almost 80% of students eat vegetables and fruit twice per day. These eating habits ought to be encouraged. The traditional Chinese diet contains plenty of vegetables and is rice-based. The present study reported a high proportion of Chinese students eat breakfast daily. In contrast, a dietary survey of young Japanese subjects revealed a low rate of individuals engaged in regular eating patterns [ 12 ]. The skipping of breakfast has been associated with lower nutritional status and the risk of cardiovascular diseases [ 13 ]. It has also been reported that less adequate breakfast habits may contribute to the appearance and further development of obesity [ 14 ]. Therefore the importance of regular eating patterns cannot be overemphasized in nutritional education. Our results showed that body figure perception was significantly different between female and male students. A number of researchers have investigated the relationship of body image and gender role. Women tend to desire a thinner figure, express more anxiety about becoming fat, and are more likely to diet than men [ 15 , 16 ]. In contrast, men have reported a desire for a heavier physique and muscularity [ 17 ]. In recent years, eating disorders have been increasing dramatically among young women. The results of our study did not confirm this suggestion to the level of statistical significance; however, it is worth pointing out that 65.0% of female students with BMI<20, which is under to normal weight range, indicated a desire to be thin. Dissatisfaction with body figure and eating disorders are closely related [ 18 - 20 ]. Being young, female, and dieting are identified risk factors that have been reliably linked to the development of eating disorders [ 21 ]. It was speculated that some of the students who were preoccupied with a thin body may develop eating disturbances. Thus, the promotion of healthy weight management practices should be considered when developing health education programs. Conclusions In conclusion, our findings reveal that the majority of students were classified into the normal BMI group, with the prevalence of BMI >30 obesity being very low in this study sample. Young female students had a greater desire to be thinner than male students. Habits involving regular eating patterns and vegetable intake were found and represent practices that ought to be encouraged. The meal and snack patterns in Chinese students were very similar to the traditional eating pattern model, although diets are changing rapidly in China and other low-income countries. The university and college arenas represent the final opportunity for nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition, especially with respect to information relating to sources of nutrition and healthy weight management. Furthermore, public demand for health and nutritional information should be taken into consideration when implementing strategies aimed at improving the nutritional well-being of individuals. Authors' contributions R.S carried out questionnaire design, manuscript drafting and total coordination of the study. K.T has been involved in drafting and revision of the article. R.A contributed to the data entry and its analysis. L.CJ contributed to the questionnaire design, data collection and language translations. N.S contributed to final approval of the manuscript. Supplementary Material Additional File 1 Table 2 containing the results of questions related to lifestyle practices with special reference to food habit. The meal patterns, consumption of fruits and vegetables, consumption of fried foods, consumption of alcohol were assessed in male and female students. The Chi-square analyses were employed to compare the behavioral differences by gender. The evaluations of statistical significance were made at the p < 0.05. Click here for file Additional File 2 Table 3 contains the results of body shape perception and health consciousness of male and female students. Male and female respondents were categorized in to 4 groups respectively, according to mean BMI of ± 1 standard deviation (SD). Analyses were made between BMI groups using Chi-square analysis. The evaluations of statistical significance were made at the p < 0.05. Click here for file | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553986.xml |
555850 | Immunogenetics of Hashimoto's thyroiditis | Hashimoto's thyroiditis (HT) is an organ-specific T-cell mediated disease. It is a complex disease, with a strong genetic component. To date, significant progress has been made towards the identification and functional characterization of HT susceptibility genes. In this review, we will summarize the recent advances in our understanding of the genetic input to the pathogenesis of HT. | Introduction Hashimoto's thyroiditis (HT) is one of the most common human autoimmune diseases responsible for considerable morbidity in women [ 1 ]. It is an organ-specific T-cell mediated disease that affects the thyroid, and genetics play a contributory role in its complexity. To date, significant progress has been made in identifying and characterizing those genes involved in the disease. In this review, we will summarize recent advances in our understanding of the genetic contribution to the pathogenesis of HT. Epidemiology and clinical features of Hashimoto's thyroiditis Goitrous autoimmune thyroiditis, or Hashimoto's thyroiditis is a common form of chronic autoimmune thyroid disease (AITD). The disorder affects up to 2% of the general population [ 2 ] and is more common in older women and ten times more frequent in women than in men [ 3 ]. In the NHALES III study, performed in the USA, the prevalence of subclinical and clinical hypothyroidism was 4.6% and 0.3% respectively [ 4 ]. Another US epidemiological study, the Whickham survey, showed the prevalence of spontaneous hypothyroidism to be 1.5% in females and less than 0.1% in males [ 5 ]. These prevalence rates are similar to those reported in Japan [ 6 ] and Finland [ 7 ]. A significant proportion of patients have asymptomatic chronic autoimmune thyroiditis and 8% of woman (10% of woman over 55 years of age) and 3% of men have subclinical hypothyroidism [ 8 ]. According the data of the 20-year follow-up to the Whickham survey cohort, the risk of developing overt hypothyroidism is four times higher in women aged between 60 and 70 years than for women between 40 and 50 years of age [ 1 ]. Subclinical hypothyroidism is characterized by an increase in serum thyrotropin (TSH) whilst serum levels of thyroxine (T 4 ) and triiodothyronine (T 3 ) remain normal. The overt disease is defined by the dramatic loss of thyroid follicular cells (thyrocytes), hypothyroidism, goitre, circulating autoantibodies to two primary thyroid-specific antigens, thyroglobulin (Tg), thyroid peroxidase (TPO), and lowered concentrations of serum TSH and T 4 [ 9 ]. Histological and cytological features of HT include a dense thyroidal accumulation of lymphocytes, plasma cells and occasional multinuclear giant cells. The epithelial cells are enlarged, with a distinctive eosinophilic cytoplasm, owing to increased number of mitochondria [ 10 ]. HT has been shown to often coexist with other autoimmune diseases such as type 1 diabetes (T1D), celiac disease, rheumatoid arthritis, multiple sclerosis, vitiligo, etc [ 11 - 14 ]. HT can also be expressed as part of an autoimmune polyendocrine syndrome type 2 (APS-2), which is usually defined by the occurrence of two or more of the following: Addison's disease (always present), AITD and/or type 1 diabetes [ 15 ], in the same patient. In common with probably all autoimmune disorders, the harmful interaction between internal (genetic) and external (environmental and endogenous) factors is required to initiate Hashimoto's disease (Fig. 1 ). Environmental triggers of HT include iodine intake [ 16 , 17 ], bacterial and viral infections [ 18 , 19 ], cytokine therapy [ 20 ] and probably pregnancy [ 21 , 22 ]. The role of dietary iodine is well defined in epidemiological studies [ 23 , 24 ] and in animal models [ 25 - 27 ] and seems to be the most significant environmental factor to induce thyroiditis. Figure 1 Possible pathogenic mechanism of Hashimoto's thyroiditis. Genetically predisposed individuals could be influenced by an environmental trigger (i.e., dietary iodine, infection, pregnancy, cytokine therapy) that induces an autoimmune response against thyroid-specific antigens by infiltrating immune cells. The autoimmune process results in preferential T helper type 1 (T H1 )-mediated immune response and induction of apoptosis of thyroid cells that leads to hypothyroidism. Pathogenesis of Hashimoto's thyroiditis Autoimmunity in Hashimoto's thyroiditis The development of the autoimmune failure of the thyroid is a multistep process, requiring several genetic and environmental abnormalities to converge before full-blown disease develops (Fig. 2 ). At the onset of disease, major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APC), particularly dendritic cells, and different subclasses of macrophages, accumulate in the thyroid [ 28 , 29 ]. APC present thyroid-specific autoantigens to the naïve T cells, leading to activation and clonal expansion of the latter. Thus, the initial stage of the disease is followed by a clonal expansion phase and maturation of autoreactive T and B lymphocytes in the draining lymph nodes. Figure 2 A scheme of autoimmune events in Hashimoto's thyroiditis. In an initial stage, antigen-presenting cells (APC), mostly dendritic cell and macrophage (Mφ) derived, infiltrate the thyroid gland. The infiltration can be induced by an envinromental triggering factor (dietary iodine, toxins, virus infection, etc.) which causes insult of thyrocytes and releasing of thyroid-specific proteins. These proteins serve as a source of self-antigenic peptides that are presented on the cell surface of APC after processing. Taking up relevant autoantigens, APC travel from the thyroid to the draining lymph node. A central phase occurs in the draining lymph node in which interactions between APC, autoreactive (AR) T cells (that survive as result of dysregulation or breakage of immune tolerance) and B cells result in inducing production of thyroid autoantibodies. In the next step, antigen-producing B lymphocytes, cytotoxic T cells and macrophages infiltrate and accumulate in the thyroid through expansion of lymphocyte clones and propagation of lymphoid tissue within the thyroid gland. This process is preferentially mediated by T helper type 1 (T H1 ) cells which secrete regulatory cytokines (interleukin-12, interferon-γ and tumor necrosis factor-α). In a final stage, the generated autoreactive T cells, B cells and antibodies cause massive depletion of thyrocytes via antibody-dependent, cytokine-mediated and apoptotic mechanisms of cytotoxity that leads to hypothyroidism and Hashimoto's disease. In autoimmune thyroditis animal models, genetically determined immune defects have been suggestively linked to the breakdown of immunological self-tolerance that results in the presentation of host autoantigens and expansion of autoreactive lymphocyte clones. These immune defects are associated with the presence of particular MHC class II haplotypes, but other immune and immune regulatory genes (i.e., CTLA-4 and others) are also involved [ 30 - 32 ]. Breakdown of the immune tolerance might occur in several ways including interrupting central tolerance (e.g. deletion of autoreactive T cells in the thymus), defects in maintaining peripheral tolerance (e.g. activation-induced T-cell death and suppressing activity of regulatory T lymphocytes) and anergy (e.g. the expression of MHC class II molecules on non-professional APC). Animal models genetically predisposed to develop an autoimmune disease, and patients with AITD, showed a lack of, or a deficiency in, a subpopulation of regulatory T cells with suppressive function [ 33 - 35 ]. The mechanisms, whereby autoreactive T cells escape deletion and anergy, and become activated, remain uncertain. There is evidence that the thyroid cell itself, by "aberrantly" expressing MHC molecules, can play the role of "non-professional " APS and present disease-initiating antigen directly to the T cells [ 36 , 37 ]. The concept of aberrant MHC class II expression was supported by studies in mice. They developed a type of Graves' Disease (GD) after being injected with fibroblasts coexpressing MHC class II and the TSH receptor (TSHR). TPO antibody production was induced after injection with fibroblasts coexpressing class II molecules and TPO [ 38 , 39 ]. Iodine is a necessary component of normal thyroid hormonogenesis. Incorporation of iodine into thyrosine residues of Tg leads to the formation of mono-iodotyrosine and di-idothyrosine derivates that subsequently undergo an oxidative coupling event resulting in the producing of T 3 and T 4 . Iodine can promote antithyroid immunity in a number of ways. Several studies suggest that iodination of Tg is crucial for recognition by Tg-reactive T cells [ 40 , 41 ]. Iodine excess can affect the Tg molecule directly, creating new epitopes or exposing "cryptic" epitopes. It has been demonstrated that a highly iodinated thyroglobulin molecule is a better immunogen than Tg of low iodine content [ 41 , 42 ]. Therefore, highly iodinated Tg may facilitate antigen uptake and processing by APC. Additionally, high doses of iodine were shown to directly affect macrophages, dendritic cells, B and T lymphocytes, resulting in stimulation of macrophage myeloperoxidase activity, acceleration of the maturation of dendritic cells, increasing the number of circulating T cells and stimulating B cell immunoglobulin production [ 25 ]. Excessive amounts of iodide ion are rapidly oxidized by TPO, thereby generating excessive amounts of reactive intermediates such as hypoiodous acid and oxygen radicals. These oxidative species damage thyrocyte cell membrane by oxidation of membrane lipids and proteins causing thyrocyte necrosis [ 43 ]. The state of severe iodine deficiency itself namely leads to a lowering of thyroid autoimmunity and an immunodeficient state in autoimmune-prone BB-DP rats. This hampers the autoreactcive T-cell generation and autoantibody production [ 25 ]. A lower degree of Tg iodination also makes this molecule less antigenic [ 42 ]. An influx of dendritic cells and macrophages to the thyroid may occur as a consequence of inflammatory events in the gland. Early non-specific necrosis of thyrocytes due to toxins (i.e. iodine, etc.), and perhaps viral or bacterial infection, can attract these cells to the thyroid. Moreover, these immune cells are normal constituents of the thyroid that are able to regulate the growth and function of thyrocytes via interleukin-1 (IL-1) and IL-6-mediated pathways [ 44 ]. A central phase of HT is characterized by the recognition of presented autoantigens by the lymphocytes, followed by an apparent uncontrolled production of autoreactive CD4+ T cells, CD8+ cytotoxic T cells and immunoglobulin G (IgG) autoantibodies. Initially, the production of self-reactive cells and autoantibodies occurs in the draining lymph nodes (Fig. 2 ). Later, the lymphoid tissue often develops directly in the thyroid gland itself. This tissue is generally very well organized, with cords of anti-Tg-antibody-producing plasma cells in the periphery. It is usually non-destructive and shows a peaceful co-existence with adjacent thyrocytes. Thyroglobulin, the main protein synthesized in the thyroid, serves both in the synthesis and in the storage of thyroid hormones. Human Tg molecules contain at least four thyroid hormone synthesis sites from the iodinated tyrosine residues at positions 5, 2553, 2567 and 2746 [ 45 ]. The hormone synthesis sites and the iodine content of Tg play an important role in its autoantigenicity [ 40 ]. Tg is one of the major autoantigens in thyroid autoimmunity and serologic studies have shown that there are at least 40 antigenic epitopes on human Tg [ 16 , 46 ]. Tg-antibodies are detected in almost all patients with AITD [ 47 ]. Anti-thyroglobulin antibodies were also reported in up to 27% of normal individuals [ 48 ]. However, numerous studies have clearly shown that the epitope recognition pattern of the natural anti-Tg antibodies is differented from that of AITD-associated anti-Tg antibodies. Most studies have demonstrarted a restricted epitope recognition pattern of AITD subjects by anti-Tg antibodies, in contrast to polyclonal reactivity observed with anti-Tg antibodies from healthy individuals [ 49 , 50 ]. Human or mouse Tg immunization induces experimental autoimmune thyroiditis (EAT) in mice [ 51 ]. The EAT induction is HLA-dependent implying an interaction between the Tg molecule and the MHC glycoproteins [ 52 ]. In addition, alterations to Tg could explain interactions between genetic and environmental factors in the aetiology of HT. Thyroid peroxidase is another significant autoantigen in the thyroid of patients affected with HT and AITD. This enzyme catalyses the oxidation of iodine to an iodinating species that forms iodotyrosines in a Tg molecule and subsequently iodotyronines [ 53 ]. TPO antibodies are heterogeneous. To date, around 180 human TPO anribodies have been cloned and sequenced. This allows for the possible identification of major features of the TPO-directed antibodies repertoire during AITD. In Graves' disease patients, heavy chain VH domains of anti-TPO antibodies preferentially use D proximal IGHV1 genes. IGHV3 genes, mainly located in the middle of the immunoglobulin heavy chain gene (IGH) cluster on chromosome 15q11, characterize HT patients more frequently. A large proportion of the anti-TPO heavy chain VH domain comes about following a VDJ recombination process that uses inverted D genes [ 54 , 55 ]. Autoantibodies against other thyroid-specific antigens such as thyrotropin receptor and sodium iodide symporter were also found in serum of HT patients. However, these antibodies occur at low frequency and do not appear to contribute any diagnostic power for HT [ 56 , 57 ]. In a final, destructive step of Hashimoto's thyroiditis, the autoreactive T cells diffusely accumulate in large numbers and infiltrate thyroid parenchyma (Fig. 2 ). In the BB-DP rat model, T-helper type 1 (T H1 )-mediated mechanisms involving production of IL-12, tumor necrosis factor-α (TNF-α) and interferon-γ play a major role in the destruction of thyrocytes, rather than T H2 type mechanisms directed by IL-4 and IL-10 [ 58 ]. The infiltration of activated scavenger macrophages into the thyroid follicles, thus destroying the thyroid cells, is compatible with T H1 -mediated mechanisms [ 59 ]. Fas and Fas ligand (FasL) expression was higher in rats with lympholytic thyroiditis indicating a role of these apoptotic molecules in thyrocyte death [ 60 ]. Apoptosis in Hashimoto's thyroiditis Autoimmune responses against specific antigens are primary determinants in thyroid autoimmunity. Other molecular mechanisms including cell apoptosis may play a role in determining the opposite phenotypic outcomes of AITD such as thyroid destruction in HT and thyroid hyperplasia in GD. T-helper lymphocytes produce cytokines that influence both immune and target cells at several levels. The predominance of T H1 or T H2 cytokines might regulate thyrocyte survival through the induction of pro-apoptotic and anti-apoptotic proteins. T H1 -mediated mechanisms lead to thyrocyte depletion in Hashimoto's thyroiditis through the involvement of death receptors and cytokine-regulated apoptotic pathways [ 61 , 62 ]. The normal thyroid gland has been shown to act as an immune privileged site having carefully regulated mechanisms of cell death and self-protection against attack by infiltrating activated T-cells induced by apoptosis [ 63 , 64 ]. Cell apoptosis occurs in the normal thyroid at a low level. As new thyrocytes are produced, old cells are destroyed in order to maintain normal thyroid volume and function. Deregulation of apoptosis, which is weakly determined by genetic susceptibility, can lead to destructive processes. Initiation of an out-of-control apoptotic mechanism in thyroid cells may be caused by various non-genetic injuries that affect expression of apoptosis inhibitor molecule Bcl-2 or membrane ligand FasL [ 65 ]. Thyrocytes from HT thyroid glands are able to hyperproduce Fas and FasL on their surfaces thus inducing fratricide apoptosis [ 66 ]. IL-1β, abundantly produced in HT glands, induces Fas expression in normal thyrocytes, the cross-linking of Fas resulting in massive thyrocyte apoptosis. This can play a role in the progression of Hashimoto's thyroiditis [ 67 ]. Immune-mediated apoptosis of thyrocytes is directed by CD8+ cells. Receptors on the target cell are triggered by lymphocyte ligands and/or released soluble factors are delivered to the target cell [ 68 ]. Receptors involved in immune-mediated apoptosis include the TNF R1 receptor, the Fas receptor and death receptors DR3 and DR4, whereas soluble mediators include substances such as perforines and TNF [ 68 - 70 ]. The common apoptotic pathway consists of subsequent activation of specific intracellular proteases known as caspases. These caspases are themselves activated by specific proteolytic cleavage or may be activated by cleavage performed by other caspases. The caspase cascade ultimately induces enzymes that progressively destroy the cell and its genetic material, finally lead to cell death. The apoptosis, or programmed cell death, can be initiated by binding death ligands, such as TNF, TNF-related apoptosis-induced ligand (TRAIL) and FasL, to the cell surface. This in turn starts intracellular signal cascading of caspases [ 71 ]. Several apoptosis signalling pathways, initiated by molecules such as FasL and TRAIL, have been shown to be active in thyrocytes and may be involved in destructive thyroiditis [ 72 ]. Fas-mediated apoptosis seems to be a general mechanism of cell destruction in AITD. In GD patients, reduced levels of Fas/FasL and increased levels of antiapoptotic molecule Bcl-2 favour thyroid cell survival and apoptosis of infiltrating lymphocytes. In contrast, the regulation of Fas/FasL/Bcl-2 expression in HT can promote thyrocyte apoptosis through homophylic Fas-FasL interactions and a gradual reduction in thyrocyte numbers leading to hypothyroidism [ 61 ]. Thus, the rate of thyrocyte apoptosis dictates the clinical outcome of thyroid autoimmunity. Though rare in normal thyroid, it markedly increases during HT, but not in GD. Therefore, regulation of thyrocyte survival is a crucial pathogenic determinant. Genetics of Hashimoto's thyroiditis Evidence for genetic susceptibility to Hashimoto's thyroiditis Abundant epidemiologic data (population-based and family-based studies, twin studies) suggest a strong genetic contribution to the development of HT. The disease clusters in families [ 22 , 73 ]. Thyroid abnormalities with clinical outcomes were observed in 33% of offspring of patients with HT or GD [ 73 ]. The sibling risk ratio (λ S ), that is the ratio of the prevalence of disease in siblings to the prevalence in the general population, can be used as a quantitative measure of the genetic contribution to the disease. Usually, a λ S of more than five indicate a significant genetic contribution to the disease development. Based on historical data, the λ S for AITD is estimated to be greater than 10, supporting a strong case of genetic influence on disease development [ 74 ]. Using HT prevalence data from the NHAHES III study, an estimated λ S value is about 28 for HT [ 74 ]. In Danish twin study, the concordance rates for Hashimoto's disease were 38% for monozygotic (MZ) twins and 0 for dizygotic (DZ) twins [ 75 ]. For HT, a recent twin study in California confirmed these results, showing concordance rates of 55% and 0% in MZ and DZ twins, respectively [ 76 ]. For thyroid antibodies, the concordance rate in the Danish twin study was twice high in MZ twins (80%) than that in DZ twins [ 75 ]. In a recent twin study in the UK, the concordance rates for Tg-antibodies were 59% and 23% in in MZ and DZ twins, respectively [ 77 ]. In this study, the concordance rates for TPO-autoantibodies were 47% and 29% in MZ and DZ twins, respectively [ 77 ]. These data suggest that HT and other AITD outcomes such as antibody production against thyroid-specific antigens have a substantial inherited susceptibility. HT seems to be a polygenic disease with a complex mode of inheritance. Immunomodulatory genes are expected to play an important role in predisposing and modulating the pathogenesis of Hashimoto's thyroiditis. Animal models of autoimmune thyroiditis Animal models of AITD still hold immense promise for the discovery of pathways, genes and environmental factors that determine the development of thyroid autoimmunity. Animals affected by experimental autoimmune thyroiditis (EAT) provide a unique opportunity to uncover disease-associated pathways, which are complicated to define in man. One of the oldest inbred models is the obese strain chicken (OS), which develops goitrous lympholytic thyroiditis with the subsequent atrophic lympholytic thyroiditis followed by a rapid onset of hypothyroidism [ 78 ]. The biobreeding diabetes-prone (BB-DP) rat expresses a form of focal lympholytic thyroiditis that under normal conditions does not lead to hypothyroidism [ 79 ]. The nonobese diabetes (NOD) mouse strain NOD-H2 h4 spontaneously develops iodine-induced autoimmune thyroiditis but not diabetes [ 26 ]. In particular, this murine strain has been extensively used to evaluate the role of iodine in the development of autoimmune thyroiditis [ 16 ]. EAT can be induced in mice by injecting with murine or human Tg, [ 80 ] and in normal syngenic recipients it is induced by the adoptive transfer of in vitro activated T cells from Tg-immunized mice [ 81 ]. The induced disease is characterized by the production of murine Tg-specific antibodies and infiltration of the thyroid by lymphocytes and other monocytes, with murine or human Tg-specific CD4+ T cells as the primary effector cells [ 80 , 82 ]. Clinical features of EAT induced in the animal models mentioned above are similar to those of human HT. For example, autoimmune thyroiditis in the NOD-H2 h4 mouse is induced by dietary iodine that supports epidemiologic data on human populations. In addition, the iodinified mouse represents high levels of IgG2b that is similar to HT patients expressing the predominance of IgG2 subclass, the human analog of murine IgG2b [ 83 ]. IgM class generally restricts Tg-antibodies of normal individuals and mice, while HT individuals and affected mice commonly produce Tg-antibodies of the IgG isotype [ 17 ]. However, anti-TPO antibodies generally detectable in HT patients could not be found in NOD-H2 h4 mice. Despite some differences between EAT and HT, these animal models have greatly contributed to the knowledge concerning the etiology and the pathogenesis of thyroid autoimmunity, most notably on the events occurring in the very early prodromal phases. Major Histocompatibility Complex (MHC) molecules are thought to play an important role in the initial stages of the development of HT and AITD. MHC molecules, or Human Leukocyte Antigen (HLA) homologs, play a pivotal role in T-cell repertoire selection in the thymus and in antigen presentation in the periphery. Crystal structures of MHC molecules show a peptide-binding cleft containing the variable region of these molecules. Genetic polymorphism of the MHC molecule determines the specificity and affinity of peptide binding and T-cell recognition. Therefore, polymorphisms within MHC class I and class II loci can play a significant role in predisposition to autoimmune disease [ 84 ]. A role of selected HLA class II genes susceptible to HT has been significantly clarified using transgenic NOD (H2A g7 ) class II-knockout mice with EAT as a model for HT [ 85 , 86 ]. In mouse genome, the H2 class II locus is homologous to the human HLA class II region [ 51 ]. A role for HLA-DRB1 polymorphism as a determining factor in HT-susceptibility, with DR3-directed predisposition and DR2-mediated resistance to the disease, was demonstrated using H2 class II-negative mice injected with HLA-DRA/DRB1*0301 (DR3) and HLA-DRB1*1502 (DR2) transgenes [ 85 ]. A role for HLA-DQ polymorphism was shown with human thyroglobulin-induced EAT in HLA-DQ*0301/DQB1*0302 (DQ8), but not HLA-DQ*0103/DQB1*0601 (DQ6), transgenic mice [ 52 ]. In summary, DR3 and DQ8 alleles are found to be susceptible, whereas DR2, DR4 and DQ6 alleles are resistant [ 30 , 87 ]. Studies on EAT-developing mice showed the differential effects of class II molecules on EAT induction. Susceptibility can be determined when class II molecules from a single locus, H2A or HLA-DQ, are examined in transgenic mice, but the overall effect may depend upon the presence of both class II molecules H2A and H2E in mice and HLA-DQ and HLA-DR in humans [ 88 ]. Polymorphism within DQ alleles can determine predisposition to HT while DRB1 molecules associated with susceptibility to HT may appear to play a permissive role. The combination of susceptibility-inducing HLA-DQ and permissive DR alleles is responsible for the association of the HLA class II region with the disease. T cells recognize an antigenic peptide via interaction of their membrane T cell receptors (TcR) with antigen-MHC complexes presented on the surface of APC. Biased or restricted TcR gene use has been reported in a variety of human or murine autoimmune diseases [ 89 ]. Biased TcR V gene in intrathyroidal T cells was also observed in mice with spontatenous (NOD strain) or human Tg-induced (CBA/J strain) thyroiditis. This confirms the primary role played by T cells in initiating EAT and the phenomenon of oligoclonal expansion of intrathyroidal T lymphocytes in early thyroiditis [ 90 ]. Sequencing of amplified TCR V beta cDNA showed that within each NOD thyroid sample at least one of the overexpressed V beta gene families was clonally expanded. For example, in the CBA/J mouse immunized with human Tg, clonally expressed T cells were shown to primarily express the murine TcR Vβ1 and Vβ13 sequences [ 91 ]. A new murine model that developed destructive thyroiditis with histological and clinical features comparable with human HT has been recently reported [ 92 ]. The transgenic mice express the TcR of the self-reactive T-cell clone derived from a patient with autoimmune thyroiditis. The T-cell clone is specific for the autoantigen thyroid peroxidase (TPO) peptide comprising amino acid residues at positions 535–551 (TPO 535–551 ) of the TPO amino acid sequence. This includes a cryptic epitope (TPO 536–547 ) preferentially displayed after endogenous processing during inflammation [ 93 ]. These results underline the pathogenic role of autoreactive human T cells and the potential significance of recognition of cryptic epitopes in target molecules such as TPO for inducing thyroid-specific autoimmune response. The two-signal theory for T cell activation requires TcR engagement of its cognate antigen-MHC complex and CD28 binding to B7 ligands (B7-1 and B7-2) on APC. Activation of T cells results in increased expression of the cytotoxic T cell antigen-4 (CTLA-4) molecule that shares homology with CD28. Although B7-1 (CD80) and B7-2 (CD86) expressed on APC can bind to both CD28 and CTLA-4 (CD152), because of higher affinity, they preferentially bind to CTLA-4 on activated T cells and attenuate the T cell response [ 94 ]. The importance of CTLA-4 in the down-regulation of T cell responses and in the induction of anergy and tolerance to alloantigens, tumors and pathogens, has been clearly demonstrated in experiments with CTLA-4 deficient mice. The mice developed a severe inflammatory disorder due to up-regulated proliferation of T cells [ 95 , 96 ]. CTLA-4 can down-regulate T cell responses involving binding and sequestering B7 molecules from CD28, therefore preventing CD28-mediated co-stimulation. Another possibility is that CTLA-4 through its intracellular domain could actively transmit a negative signal resulting in down-regulation of activated T cells [ 97 ]. The crucial role of CTLA-4 in maintaining self-tolerance breakdown of which leads to the initiatition of a primary autoimmune response has been demonstrated in several murine models of autoimmune diabetes [ 98 ] and autoimmune thyroiditis [ 32 ]. Human Leukocyte Antigen class I and II genes Genes of the human MHC region are clustered on chromosome 6p21 and encode HLA glycoproteins and a number of additional proteins, which are predominantly related to immune response. The MHC locus itself contains three groups of genes: class I genes encoding HLA antigens A, B and C, class II genes encoding HLA-DR, DP and DQ molecules and class III genes [ 99 ]. Previous studies in the early 1980s investigated the HLA locus in relation to the genetics of HT. Associations between HLA and HT have both been analysed by serologic typing of HLA and DNA typing using sequence-specific oligonucleotide probe analysis or restriction fragment length polymorphism. In Asians, HLA class I (A2, B16, B35, B46, B51, B54, C3) and HLA class II (DR2, DR9, DR53, DQ4) genes showed an association with the disease [ 31 , 100 - 105 ]. In Caucasians, HT is associated with HLA class II genes such as DR3, DR4, DR5, DQA1*0301, DQB1*0201 and DQB1*0301 [ 106 - 120 ] but not with the HLA-DP and HLA class I (HLA-A, HLA-B and HLA-C) genes [ 113 , 114 , 121 ]. However, some studies could not reveal an association between HLA-DQ and DR genes and Hashimoto thyroiditis [ 114 , 122 , 123 ]. Reports of disease-associated alleles are not consistent, but associations appear to be strongest with alleles in the HLA-DR and -DQ loci. This has also been suggested by studies in transgenic mouse [ 30 , 52 , 85 - 87 ]. Early linkage, non-genome-wide studies of the HLA region have failed to detect linkage between the HLA locus and HT [ 124 - 129 ]. Using dataset of 56 US Caucasian multigenerational families, genome-wide scans has revealed a susceptibility locus AITD-1 located on chromosome 6p [ 130 ]. The AITD-1 locus is common for both general forms of thyroid autoimmunity, HT and GD [ 130 ]. This locus was replicated in the expanded dataset of 102 US Caucasian families but is distinct from the HLA gene cluster [ 131 ]. Whole-genome scans of a large family with members affected with vitiligo and HT mapped a HT susceptibility locus that shared both the MHC region and the non-MHC AITD-1 [ 132 ]. However, evidence for linkage between the HLA locus and HT (or autoimmune thyroid disease) has not been confirmed by further whole-genome scans of other affected families [ 133 , 134 ], sibling pairs [ 135 ], or within HLA-DR3 positive families [ 120 ]. The lack of linkage means, for instance, the DR3 gene did not cause the familial segregation of Hashimoto's disease while a relatively strong and consistent association showed that HLA-DR3 conferred a generalized increased risk of HT in the general population. These data did not support a major role for the HLA region in the susceptibility to HT and may imply that the DR3 gene modulates the effect of other non-HLA susceptibility gene. However, a linkage between the HLA region and HT was recently shown in the data set of 40 US multiplex families affected with AITD and type 1 diabetes [ 136 ]. The linkage to HT was found to be weaker than to diabetes, suggesting that additional, non-HLA loci were contributing to the joint susceptibility to AITD and T1D. Among HLA-DR alleles, HLA-DR3 was detected as the only associated gene for Hashimoto's thyroiditis and diabetes [ 136 ]. Indeed, DR3 seems to represent the major HLA allele, which contributes to the shared susceptibility to T1D and AITD. These findings, however, need to be replicated in larger data sets because early family [ 137 , 138 ] and case-control [ 139 , 140 ] studies have not shown the unique role for HLA-DR3 allele in conferring shared susceptibility to T1D and thyroid autoimmunity. The HLA region has been established to be involved in multiple autoimmune disorders [ 141 ]. The mechanisms by which HLA molecules influence the susceptibility to autoimmune disorders become more and more clear. Different HLA alleles could have different affinities to autoantigenic peptides. Therefore, certain alleles can bind the autoantigenic peptide, with the subsequent recognition by T cells that have escaped self-tolerance, whereas others may not [ 142 ]. The possibility of certain class II alleles to bind and present thyroid-specific antigens such as TSHR or Tg peptides has been shown in vitro [ 143 ] and in mice with EAT [ 144 ]. Thyroid autoantigens need to occur in the thyroid or its draining lymph nodes in order for them to be presented by HLA molecules. It has been suggested that an aberrant intrathyroidal expression of MHC class II molecules by thyrocytes is necessary to initiate thyroid autoimmunity [ 145 , 146 ]. This hypothesis is supported by detection of the expression of HLA class II molecules by thyroid epithelial cells in HT and GD patients [ 147 , 148 ] and in studies on animal models with experimentally induced thyroid autoimmunity [ 85 , 145 , 149 , 150 ]. The aberrant expression of HLA class II antigen by thyrocytes can initiate autoimmune responses through direct thyroid self-antigen presentation or a secondary event following on from cytokine secretion by infiltrated T lymphocytes [ 148 , 151 ]. Genetic contribution of HLA varies depending on the disease. HLA involvement in T1D, rheumatoid arthritis or multiple sclerosis is large and can constitute more than 50% of the genetic risk [ 84 , 152 ]. Contributions of HLA alleles as genetic risk factors to HT are much weaker [ 118 , 153 ]. HLA class I and II genes appear to contribute to the autoimmunity in general but not to organ specificity. Their role in the predisposition to HT is rather non-specific [ 62 , 117 ]. The HLA class I and II genes appear not to be the primary HT genes, and are likely to be modulating genes that increase the risk for AITD contribution by other genes. HLA class III and other non-HLA genes, located in the HLA region, are also critical to the immune response. It is possible that HLA associations as seen in thyroid autoimmunity are due partially to genetic variation in these closely linked immune regulatory genes and their linkage disequilibrium with class I and II genes [ 154 ]. HLA class III genes and non-HLA genes of the HLA region The HLA class III region lies between class I and II genes and encodes important immunoregulatory proteins such as cytokines [tumour necrosis factor (TNF), lymphotoxin alpha (LT-α) and beta (LT-β)], complement components (C2, C4, properdin factor B) and heat shock proteins (HSP) [ 155 ]. Both TNF and LT-α mediate B-cell proliferation and humoral immune responses [ 154 ]. TNF has been found to enhance cellular expression of HLA class I and II antigens, and enhances adhesion and complement regulatory molecules in the thyroid gland of HT patients. Alterations to the above could promote the autoimmune process [ 156 ]. However, case-control studies showed no association between polymorphisms within the TNF and LT-α genes and HT in Germans [ 112 ], UK Caucasians [ 118 ] and Koreans [ 157 ]. HSP70 gene cluster consists of three genes encoding HSP70-1, HSP70-2 and HSP-Hom proteins. They are expressed in response to heat shock and a variety of other stress stimuli (e.g. oxidative free radicals, toxic metal ions and metabolic stress). HSPs are also important for antigen processing and presentation [ 158 ]. Genetic variations within all three HSP70 genes were tested in British patients with HT and no associations were found [ 118 ]. Polymorphisms of complement component-encoding genes have not yet been evaluated in relation to HT. Meanwhile, finding a link between frequency disturbances in BI and C4A allotypes and one of the forms of thyroid autoimmunity, postpartum thyroiditis [ 159 ], may be an intriguing future study in HT patients. Other genes crucial to the immune response, including TAP (transporters associated with antigen processing), LMP (large multifunctional protease), DMA and DMB genes are located within the HLA class II region [ 155 ]. Protein products of TAP (TAP1 and TAP2) and LMP2 (LMP2 and LMP7) genes participate in the proteolysis of endogenous cytoplasmic proteins into small fragments and subsequent transportation of these self-peptides from the cytoplasm into the endoplasmic reticulum, the site of HLA class I assembly [ 160 ]. To date, one investigation has been concerned with the association between TAP1 and TAP2 genes and Hashimoto's thyroiditis. No significant association was observed in the British population [ 118 ]. The genetic role of LMP in HT has not yet been examined. An association between the R60 allele of the LMP2 gene and GD was observed [ 161 ]. Additionally, quantitative defects in the amount of transcription products of TAP1, TAP2, LMP2 and LMP7 genes were found in lymphocytes of patients with AITD [ 160 ]. These findings suggest that defective transcription of HLA class I-processing genes could contribute to the quantitative defect in cell-surface expression in autoimmune lymphocytes in HT. Further evaluation of the role of such class I-processing genes as TAP and LMP is necessary. DMA and DMB genes are involved in the assembly of HLA class II peptides. These genes encode subunits of a functional heterodimer that is critical for class II antigen presentation [ 160 , 162 ]. Based on nucleotide variation within exon 3, three rare DMB alleles (DMB*01kv1, DMB*01kv2 and DMB*01kv3) have been detected in Korean HT patients while these DMB variants have not been found in healthy subjects [ 163 ]. However, these DMB alleles have not yet been functionally characterized. In summary, there is a significant dearth of information on how HLA class III genes and non-HLA genes, located in the HLA region, contribute to the pathogenesis of HT. Further studies are required to clarify the involvement of these genes in HT susceptibility. CTLA-4 gene The CTLA-4 gene is the most frequently studied of the immune modulatory genes located outside the HLA region, in relation to the genetics of HT. This gene encodes a costimulatory molecule, which suppresses T-mediated immune response and is crucial in the maintenance of peripheral immunological self-tolerance [ 164 ]. An inventory of case-control studies based on the association between three polymorphic markers within the CTLA-4 gene [A49G dimorphism in the leader peptide, C (-318) T substitution in the promoter region and a dinucleotide repeat polymorphism at the 3'-untranslated region (3'-UTR)] and HT is reviewed in [ 165 ]. Results of these studies, except for those for the C (-318) T single nucleotide polymorphism, suggest that polymorphisms within the CTLA-4 gene are associated with the development of HT. Family studies showed linkage between CTLA-4 and GD [ 166 ], thyroid antibody production [ 167 ] and autoimmune thyroid disease [ 12 , 62 ] but not specifically to HT, probably due to lack of their power [ 129 , 130 , 135 ]. Classical linkage analysis is suitable for detecting susceptibility loci with major genetic effects. CTLA-4 demonstrates a modest but significant effect in the genetics of HT. To detect a locus with a modest genetic effect, a large number (at least 400) of affected families should be tested [ 168 ]. This investigation has recently been performed involving about 600 AITD families and more than 1300 affected patients [ 169 ]. The CTLA-4 gene has been found to play a critical role in the pathogenesis of autoimmune diseases such as GD, HT and T1D [ 62 , 153 , 169 ]. Disease susceptibility was mapped in the 6.1-kb 3' untranslating region of CTLA-4. Allelic variation was correlated to altered mRNA levels of soluble form of CTLA-4 [ 169 ]. This alternative splice form of CTLA-4 lacks exon 3 encoding the transmembrane domain but maintains exon 2 encoding the ligand-binding domain [ 170 ]. The short form of CTLA-4 can bind CD80/86 and inhibit T-cell proliferation [ 171 ]. The soluble CTLA-4 (sCTLA-4) is expressed constitutively by T regulatory cells suppressing the effector T-cell response [ 172 ]. Its role in autoimmune disease is not exactly clear, but sCTLA-4 was observed significantly more often in patients with AITD [ 173 ] and myasthenia gravis [ 174 ] in comparison with non-affected subjects. Patients with AITD and myasthenia gravis had an aberrant expression of the CTLA-4 products, with high levels of sCTLA-4 and low levels of the intracellular form [ 175 ]. Soluble CTLA-4 might play an important role in immune regulation by binding with the B7 molecules, thus interfering with the binding of CD28 and/or full-length CTLA-4. Interference of sCTLA-4 with B7/CTLA-4 interactions could block suppressive signals transferred via surface-bound CTLA-4. Therefore, high concentrations of sCTLA-4 in serum might contribute to disease manifestations through interference of sCTLA-4 with B7/CTLA-4 interaction. It may be that the amino acid change at codon 17 of the signal peptide could alter the function of the signal peptide to direct intracellulat trafficking of CTLA-4. In in vitro expreriments, the Ala17 (G49) allele was found to represent a translation product, which was not glycosylated in one of two N-linked glycosylation sites [ 176 ]. This aberrantly glycosylated product was shown to be further translocated from the endoplasmic reticulum back to cytoplasm and, probably, to become a target for proteolytic degradation. In addition, the distribution of Ala17 CTLA-4 variant on the surface of COS1 cells is significantly less density than the Thr17 variant of CTLA-4 [ 176 ]. These fundings suggest that the Ala17 allele is linked to the inefficient glycosylation of CTLA-4, which subsequently could affect suppressing effects of the CTLA-4 molecule. This could also explain observations showing that the G49 allele of the CTLA-4 signal peptide is associated with accelerated proliferation of T lymphocytes in human subjects homozygous for this allele, and with suppression of the downregulation of T-cell activation in response to IL-2 [ 177 , 178 ]. The codon 17 single nucleotide polymorphism (SNP) is shown to be in tight linkage disequilibrium with another SNP situated at position (-318) of the CTLA-4 promoter region and with the (AT) n repeat polymorphism at the 3'-UTR of the CTLA-4 gene [ 176 , 179 - 181 ]. For the C (-318) T SNP, the protective T (-318) allele demonstrated higher promoter activity than the alternative C allele in a luciferase expression assay [ 182 ]. Since the (-318) dimorphism occurs in a potential regulatory region, this suggets that this nucleotide substitution may influence the expression of CTLA-4. However, this possibility remains to be explored. The (AT) n repeat polymorphism at the 3'-UTR of the CTLA-4 gene has been shown to affect the expression of this costimulatory molecule [ 174 ]. Adenylate- and uridylate-rich elements (AUREs) presented in the 3'-UTRs can regulate stability of eukaryotic mRNAs, and their presence correlates with rapid RNA turnover and translational and posttranslational control [ 183 , 184 ]. The AT repeats in the 3'-UTR of CTLA-4 might represent a special type of of AUREs. CTLA-4 mRNA with longer (AT) n alleles have shorter half-lives and, hence, are more unstable [ 174 , 185 ]. Indeed, the (AT) n microsatellite in the 3'-UTR influences the mRNA stability. Additionally, the CTLA-4 AT-repeat polymorphism was recently shown to alter the inhibitory function of CTLA-4. The long AT-repeat allele is associated with reduced control of T-cell proliferation and thus contributes to the pathogenesis of GD [ 186 ]. The AT-repeat may also affect splicing of one or more of the alternative CTLA-4 transcripts but this should be clarified. Ueda et al . [ 169 ] showed that another polymorphism (A6230G, or CT60 SNP) located in the first position of the 3'-UTR correlates with higher expression of a soluble CTLA-4. In this study, the highest power of linkage with GD was found for this SNP and three other SNPs (JO27, JO30 and JO31) within a 6.1-kb segment of the 3'-UTR, but not for the (AT) n repeat polymorphism [ 169 ]. However, no T-cell function data were presented. Thus, further investigations are necessary to evaluate functional significance of these SNPs. Due to the linkage disequilibrium, it is currently not possible to determine whether one, or both, are of physiological importance. It can not be excluded that allele combination of several closely linked CTLA-4 polymorphisms might form a functionally significant haplotype that is directly involved in the susceptibility to autoimmune disease [ 187 , 188 ]. It should be noted that the genomic region 2q33 linked to autoimmune disease contains cluster of three genes encoding costimulatory molecules CTLA-4, CD28 and inducible costimulator (ICOS) [ 189 ]. However, genetic studies showed that the AITD gene in the 2q33 locus is the CTLA-4 gene and not the CD28 or ICOS genes [ 167 , 169 , 181 ]. The CTLA4 gene should be recognised as the first major known non-HLA locus of human autoimmunity and that its role in the pathogenesis of HT is rather general and non-specific [ 74 , 153 ]. Association of CTLA-4 with the production of thyroid antibodies [ 167 , 190 ], an event that often represents the subclinical stage of AITD [ 1 ], can explain non-specific mechanism of CTLA-4-mediated susceptibility to the development of thyroid autoimmunity. The association of the CTLA-4 gene with several autoimmune diseases such as T1D [ 153 , 169 ], Addison's disease [ 191 , 192 ], multiple sclerosis [ 193 , 194 ], myasthenia gravis [ 175 ] and all clinical outcomes of AITD [ 74 ], can also explain the general contribution of CTLA-4 to autoimmunity. Interestingly, AITD, Addison's disease and autoimmune diabetes frequently coexist in patients with the autoimmune polyendocrine syndrome type II as mentioned above. The above disorders seem to share a genetic background, and CTLA-4 could represent a common susceptibility focus for them [ 195 ] Other immune regulatory genes In initial phases of AITD, oligoclonal expansion of T lymphocytes occurs in the thyroid gland. These T cells are restricted by their T cell receptor V gene use [ 89 , 90 ]. Therefore, the TcR may be considered a likely candidate gene for AITD and HT. Early case-control investigations showed a lack of association between HT and the T-cell receptor-α gene in the US white population [ 111 ] but not the T-cell receptor-β gene in the Japanese [ 102 ]. Linkage analysis using a US Caucasian AITD family dataset [ 129 ] and Tunisian affected pedigree [ 196 ] has eliminated the T-cell receptor V alpha and V beta gene complexes, located on 14q11 and 7q35, respectively, as candidate genes for susceptibility to thyroid autoimmunity. Therefore, the TcR genes are not major susceptibility genes for HT and AITD. Another likely candidate among immune-related genes was the IGH gene because HT individuals commonly produce Tg-autoantibodies restricted by IgG class [ 50 ]. Early investigations found an association between IgH Gm allotypes and AITD in the Japanese [ 197 , 198 ]. However, these findings have not been confirmed in Caucasians [ 129 , 196 ]. Cytokines are crucial in the regulation of immune and inflammatory responses. Multiple investigations showed the important role of these regulatory molecules in directing autoimmune and apoptotic pathogenic processes, of particular, in central and late stages of the development of HT [ 72 , 80 , 199 ]. Therefore, cytokine genes might be good candidates for HT. Intrathyroidal inflammatory cells and thyroid follicular cells produce a variety of cytokines, including interleukin-1α (IL-1α), IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-14, tumor necrosis factor-α, and interferon-γ [ 200 ]. Hunt et al . [ 201 ] evaluated 15 polymorphisms within nine cytokine genes for IL-1α, IL-1β, IL-1 receptor antagonist (IL1RN), IL-1 receptor 1, IL-4, IL-4 receptor, IL-6, IL-10, and transforming growth factor-β in British patients with AITD. They only found a significant association for one of those. The T-allele of the IL-4 promoter [T (-590) C] polymorphism was associated with lower risk of GD and AITD but not HT [ 201 ]. Blakemore et al. [ 202 ] failed to find an association between a polymorphic minisatellite in the IL1RN gene and HT in another group of affected patients from UK. Thus, it may be concluded that these genes are not major susceptibility genes for thyroid autoimmunity but need to be further studied. The autoimmune regulator (AIRE1) gene is known to contribute to the pathogenesis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare monogenic autoimmune disease with endocrine components including T1D, adrenal failure, and thyroid dysfunction, with major autoantibodies directed against adrenal, pancreas, and thyroid tissue [ 203 ]. However, studies in UK patients showed no relation between a 13-bp deletion at nucleotide 964 in exon 8 (964del13) of the AIRE1 gene, a common disease-associated marker for APECED in British population, and HT [ 204 ]. The vitamin D-mediated endocrine system plays a role in the regulation of calcium homeostasis, cell proliferation and (auto) immunity. 1,25-Dihydroxi-vitamin D 3 (1,25(OH) 2 D 3 ) is the most active natural vitamin D metabolite that effectively prevents the development of autoimmune thyroiditis in an animal model [ 205 ] and inhibits HLA class II expression on endocrine cells [ 206 ]. C/T polymorphism located at intron 6 of the vitamin D 1α-hydroxylase (CYP1α) gene failed to show association with HT in Germans [ 207 ]. Two polymorphic markers within the vitamin D-binding protein gene encoding another member of the vitamin D metabolic pathway also showed no association with HT in the German population [ 208 ]. However, among two polymorphic sites tested at the vitamin D receptor (VDR) gene, the Fok I(+) allele of the FokI/restriction fragment length polymorphism was found to be associated with higher risk HT in Japanese females [ 209 ]. Meanwhile, the VDR gene remains to be a likely candidate for the common autoimmune susceptibility gene because it has been found to be associated with autoimmune disorders such as GD [ 210 ], Addison's disease [ 211 ], multiple sclerosis [ 212 ] and T1D [ 213 ]. Thus, a wide variety of non-HLA immune regulatory genes located outside the HLA region showed no significant linkage or association with HT and AITD except for the CTLA4 gene. However, we still cannot estimate whether or not these genes significantly contribute to HT susceptibility due to a serious shortfall in information about their role in this disorder. It cannot be excluded that other genes in linkage disequilibrium with these genes are the susceptibility genes at these loci. Thyroid-specific genes Antibodies against thyroid peroxidase are one of the most specific features of HT [ 214 ]. Therefore, the TPO gene is expected to be a putative candidate responsible not only for susceptibility to HT but also for specific determination between two common outcomes of AITD, such as HT and GD. Genetic transmission of the recognition by antibody of the TPO immunodominant region and the TPO B domain has been described in families affected with HT [ 215 ]. This transmission could be explained by genetic variations within the TPO gene. However, case-control studies showed lack of association between the TPO gene polymorphisms and AITD [ 113 , 216 ]. These data suggest that the thyroid peroxidase gene does not play an important role in predisposition to HT. Subsequent studies are necessary to clarify exactly whether this gene is a true susceptibility gene for AITD. Within the other thyroid-specific gene, the TSHR gene, the T52P amino acid substitute was examined in US white and Thai populations but no association with HT was found [ 217 , 218 ]. Various genome-wide scans have failed to detect linkage between the thyrotropin receptor gene and HT or AITD [ 130 , 133 - 135 , 219 , 220 ]. However, two microsatellites, an (AT) n marker at intron 2 of the TSHR gene and a (CA) n marker that was mapped to approximately 600 kb of the TSHR gene, have been shown to be strongly associated with HT in Japanese patients [ 221 , 222 ]. The TSHR gene, therefore, does not seem to be a major susceptibility gene for HT, although a minor role cannot be excluded. Tg-specific autoantibodies are common in AITD. The thyroglobulin gene makes a significant contribution to HT and AITD. Whole-genome scans in Japanese-affected sibling pairs have detected a HT susceptibility locus on chromosome 8q24, with a maximum linkage to marker D8S272 [ 135 ]. This marker is separated by 4.6 megabases (Mb) from the Tg gene. Subsequent studies of the mixed US and European Caucasian family dataset has confirmed the susceptibility locus to be on chromosome 8q24, with the maximum linkage to markers D8S514 and D8S284 [ 74 , 131 , 223 ]. These markers border a large region of chromosome 8 spanning about 15 Mb. The thyroglobulin gene is located within this region. Moreover, a new microsatellite marker Tgms2 inside intron 27 of the Tg gene showed strong evidence of linkage and association with AITD [ 223 , 224 ]. Two new microsatellites have recently been described in introns 29 and 30 of the thyroglobulin gene that can be useful for further linkage studies in families with autoimmune thyroid diseases [ 225 ]. Using a high-density panel of SNPs within the human and murine Tg genes, Ban et al . [ 226 ] identified a unique SNP haplotype, consisting of an exon 10–12 SNP cluster in both genes and, additionally, exon 33 SNP in the human gene, associated with AITD in humans and with EAT in mice. Taken together, these data strongly suggest that the thyroglobulin gene could represent the susceptibility gene for HT and AITD on 8q24 [ 74 , 227 ] and, therefore, be characterized as the first thyroid-specific susceptibility gene for thyroid autoimmunity [ 228 ]. The Tg gene spanning over 300 kilobases long is expected to harbour more than one haplotype block associated with AITD since the length of a linkage disequilibrium block of SNPs is shown to be less than 100 kilobases [ 229 ]. It seems that this gene is AITD-specific but is not a HT-specific susceptibility gene. The manner in which the Tg gene can be a predisposition to AITD remains unclear. It could be that amino acid variations within the Tg gene can affect the immunogenicity of Tg. The evidence that iodination of thyroglobulin affects its immunogenicity favours this suggestion [ 230 , 231 ]. However, additional studies are required to evaluate that. Recent investigation in Tunisians showed significant association of two polymorphic microsatellites (D7S496 and D7S2459) close to the PDS gene (7q31) with GD and HT, and one of them, D7S496, was linked to GD only [ 232 ]. The PDS gene encodes a transmembrane protein known as pendrin. Pendrin is a chloride/iodide transporting protein identified in the apical membrane of the thyroid gland [ 233 ]. Data of Kacem et al . [ 232 ] suggest that the PDS gene might be considered a new susceptibility gene to autoimmune thyroid diseases, having a different involvement with different diseases. However, studies in other populations are necessary to support a role for the PDS gene in thyroid autoimmunity and HT. Finally, a role for other genes specifically expressed in the thyroid gland, has yet to be defined. These genes include those encoding thyrotropin-β, thyroid-specific factor-1, sodium iodide (Na + /I) symporter and paired box transcription factor-8, among others. They also need to be evaluated for any putative impact on HT. Apoptotosis-related genes Two polymorphic sites within the FasL gene were recently tested in HT Caucasian patients from Italy and Germany. No association between these polymorphisms and the disorder was shown [ 234 ]. Assuming a lack of association of the naturally occurring FasL gene polymorphisms with multiple other autoimmune diseases tested, we conclude that genetic variation within this gene does not contribute to autoimmunity. Inactivating mutations within the Fas and FasL genes are associated with carcinogenesis [ 235 , 236 ]. This situation is common among apoptotic-related genes encoding caspases, death receptors, decoy receptors and death ligands as well as for genes that encode other types of signalling molecules [ 237 ]. However, since apoptotic mechanisms play a critical role in pathogenesis and progression of HT, genes associated with programming cell death should be evaluated whether or not they confer susceptibility to HT. Other genes Due to the prevalence of thyroid autoimmunity in females, gender-related genes could also be considered as putative candidates for HT susceptibility. Some of these genes, such as the CYP19 gene encoding aromatase that participates in estrogen synthesis, and genes for estrogen receptor-α (ESR1) and -β (ESR2), were examined but showed no linkage with HT [ 238 ]. The ESR1 and ESR2 genes demonstrated no association with AITD in the Japanese [ 239 , 240 ]. It seems that the CYP19 and both estrogen receptor genes do not predispose to HT and AITD. Other gender-specific genes could contribute to AITD. A possible involvement of such genes has been shown for GD with the discovery of a susceptible locus on chromosome X [ 238 ]. The SEL1L gene, encoding a novel transcription factor, was recently described [ 241 ]. The gene is located on chromosome 14q24.3-q31 close to the GD-1 susceptibility locus [ 128 , 130 , 219 ] and considered a likely candidate for thyroid autoimmunity. However, a case-control study in the Japanese population detected no association of a dinucleotide (CA) n repeat polymorphism in the intron 20 of the SEL1L gene with AITD [ 242 ]. This gene may be a potentially predisposing gene to T1D because it is specifically expressed in adult pancreas and islets of Langerhans [ 241 ]. It lies in the vicinity to IDDM11, a susceptibility locus to this autoimmune disease, on chromosome 14q24.3-q31 [ 243 ]. A new zink-finger gene designated ZFAT (a novel zink-finger gene in AITD susceptibility region) has been recently found on chromosome 8q24 [ 244 ]. The T allele of the Ex9b-SNP10 dimorphism representing an adenine-to-thymidine substitution within intron 9 of this gene was shown to be associated with high risk for AITD in Japanese patients. Functional studies showed that the Ex9b-SNP10 significantly affects the expression of the small antisense transcipt of ZFAT (SAS-ZFAT) in vitro and this expression results in the decreasing expression of the truncated form of ZFAT (TR-ZFAT) [ 244 ]. This SNP is located in the 3'-UTR of TR-ZFAT and in the promoter region of SAS-ZFAT. Full-length ZFAT and TR-ZFAT encode a protein with unknown function, which has eighteen and eleven repeats of zink-finger domains, respectively. Both molecular variants of ZFAT are expressed in different tissues including peripheral blood lymphocytes, while SAS-ZFAT is exclusively expressed in peripheral blood B cells and represents a non-coding RNA with putative regulatory function [ 245 ]. The disease-associated polymorphism can play a significant role in B cell function by enfluencing the expression level of TR-ZFAT through regulation of transcription of SAS-ZFAT. Interestingly, Shirasawa et al . found no association of the thyroglobulin gene with AITD when studying different ethnic groups [ 244 ]. These results suggest that the ZFAT gene could implicate the susceptibilty to AITD on chromosome 8q24 but that it needs to be strongly replicated in other populations. Additionally, the ZFAT gene should be functionally studied to clarify whether the ZFAT or thyroglobulin gene are true contributors of genetic susceptibility to AITD and HT on 8q24. Non-defined susceptibility loci for Hashimoto's thyroiditis and autoimmune thyroid disease At present, the CTLA-4 (chromosome 2q33), thyroglobulin (or ZFAT) (8q24) and likely HLA genes (6p21.3) are the only susceptibility loci for HT and thyroid autoimmunity to be mapped. Two HT-specific susceptibility loci that have been detected in mixed Caucasian families from USA and Europe, HT-1 (13q) near marker D13S173 and HT-2 on chromosome 12q in the vicinity of marker D12S351, are still not defined [ 130 ]. HT-2 locus has been subsequently replicated in the extended dataset, with a peak linkage close to marker D12S346, which HT-1 does not have [ 223 ]. Possible candidate genes for susceptibility to HT positioned within the HT-2 locus may include the BTG1 and CRADD genes. The BTG1 gene encodes B-cell translocation protein-1, which play an immune regulatory role as a negative regulator of the proliferation of B cells [ 246 ]. The GRADD gene encodes CASP-2 and RIPK-domain-containing adaptor with death domain, that represents apoptotic function, inducing cell apoptosis via recruiting caspase 2/ICH1, TNF receptor 1, RIPK-RIP kinase and other proteins [ 247 ]. AITD-1 locus located on chromosome 6p is very close yet distinct from the HLA region [ 120 , 130 ]. It has been shown that the AITD-1 is positioned in the same location as susceptibility loci for T1D (locus IDDM15) [ 248 ] and systemic lupus erythematosus [ 249 ]. This may imply that a general autoimmunity susceptibility gene is located in this region. The AITD-1 locus contains an interesting positional candidate gene such the SOX-4 gene, encoding a transcription factor that modulates differentiation of lymphocytes [ 250 ]. A whole-genome scan in Japanese showed evidence for linkage with AITD on chromosome 5q31-q33 [ 135 , 251 ]. The 5q31 locus was replicated by recent genome-wide scan in Caucasian population, the Old Order Amish of Lancaster County, from Pennsylvania [ 220 ]. This locus harbours a cluster of cytokine genes and, therefore, several positional candidate genes occur in this region and need to be evaluated. In the Chinese, a whole-genome screening for AITD susceptibility found two chromosome regions (9q13 and 11q12) linked to AITD [ 134 ]. Susceptibility genes have yet to be defined within these regions. However, the 9q13 locus harbours a putative candidate gene such as the ANXA1 gene, whose product annexin A1 prevents the production of inflammatory mediators [ 252 ]. The 11q12 locus contains several interesting candidate genes encoding immune modulators (CD5 and CD6) and possible components of antigenic peptide processing (PSMC3) and transport (PTH2). In a large Tunisian family affected with AITD, a susceptibility locus was mapped on 2p24 [ 133 ] This locus harbors two possible candidate genes such as the FKBP1B gene, product of which demonstrates immune modulating activity [ 253 ], and the TP53I3 gene encoding p53-inducible protein 3 that is involved in p53-mediated apoptotic pathway [ 254 ]. These data suggest that both HT and AITD show genetic heterogeneity in different populations. Susceptibility loci differ in their chromosome location depending on the population being tested. The contributory value of these genes to the disease pathology varies significantly depending on the ethnic background. A gene, that has a major effect on the susceptibility to HT in one population, may contribute weakly in other population. To date, several regions of linkage to HT and AITD have been defined. Further studies are required to find a true susceptibility gene in these genomic regions to reveal the functional significance of disease-associated polymorphisms within these genes. Conclusion AITD can be initiated in individuals genetically predisposed to AITD by non-genetic (environmental) triggers such as dietary iodine, infection, pregnancy, cytokine therapy (Fig. 1 ). This interaction leads to different clinical phenotypes of thyroid autoimmunity such as Graves' disease, Hashimoto's thyroiditis or production of thyroid antibodies. HT and GD are two distinct but related clinical outcomes of AITD. It seems that both thyroid diseases have common pathogenic mechanisms as their initial steps including breakdown of the immune tolerance and accumulation of T lymphocytes in the thyroid gland. Sequence variants of CTLA-4, associated with increased levels of the soluble form of this immune costimulator and with stability of CTLA-4 mRNA, could play a crucial role in the earliest stages of AITD (i. e. breakdown of self-tolerance and surviving autoreactive T lymphocytes). This role might be sufficient to regulate subsequent steps in the development of autoimmune responses to the production of thyroid autoantobodies [ 167 ]. Environmental factors (particularly, iodine intake and infection) could cause insult of the thyrocyte followed by abnormal expression of MHC class I and class II molecules, as well as changes to genes or gene products (such as MHC class III and costimulatory molecules) needed for the thyrocyte to become an APC [ 255 ]. In this stage, a modulating role of sequence variants of HLA class II molecules could become pivotal in binding and presenting thyroid antigenic peptides derived from Tg, TPO and TSHR. Genetic variations in Tg, and probably in TSHR and other thyroid-specific genes, might be responsible for generating an autoimmune response. In later stages, thyroid autoimmunity could be switched towards GD or HT. GD is characterized by T H2 -mediated switching of thyroid-infiltrating T cells. These induce the production of stimulating anti-TSHR antibodies by B cells and anti-apoptotic mechanisms that lead to clinical hyperthyroidism. In HT, preferential T H1 response initiates apoptosis of thyroid cells and results in clinical hypothyroidism [ 22 ]. It is clear that a number of loci and genes determine genetic predisposition to HT, with varying effects. These loci could be unique to HT or general for both HT and GD. Several whole-genome scans showed results suggesting that there is significant shared susceptibility to HT and GD [ 130 , 131 , 134 , 135 ]. This is also supported by the frequent coexistance of both diseases in affected families [ 74 , 133 ]. Preliminary data suggest that shared genetic susceptibility involves both immune regulatory (i. e. CTLA-4 and HLA) and thyroid-specific genes (i.e. Tg). These genes are not responsible for the determination of pathogenic mechanisms of thyroid autoimmunity distinct for HT and GD. It remains unclear which susceptibility genes are specifically involved in the HT pathogenesis. The CD40 gene, an important immune modulator, appears to act as a GD-specific susceptibility gene. The gene is located within the 20q11 locus and shows significant linkage to GD, but not to HT, in UK Caucasians [ 74 , 130 , 131 , 256 , 257 ]. Subsequent analysis found the CD40 gene to be associated with GD [ 258 ]. However, this finding needs to be independently confirmed in other population samples. A probable susceptibility gene that could direct switching towards GD or HT is thought to be located within the 5q31 locus, which is linked to AITD and contains a cytokine gene cluster. Different sets of cytokines are known to regulate switching to T H1 or T H2 type mechanisms [ 58 ]. There may be two susceptibility genes, each of which uniquely contributes to the development of HT- or GD-specific pathogenesis. The IL-4 promoter [T(-590) C] polymorphism also appears to be associated with GD, but not with HT [ 199 ]. IL-4 mediates T H2 type mechanism, which can lead to hyperthyroidism [ 259 ]. Another HT-specific susceptibility gene(s) may be an apoptotic gene. Apoptosis of thyroid follicular cells is the hallmark of HT and might be the primary cause of death of thyrocytes compared to T cell-mediated cytotoxity [ 69 ]. Thus, it is necessary to identify additional susceptibility genes and disease-associated polymorphisms in apoptotic genes in AITD- and HT-linked loci by using a fine mapping approach and high-density panels of SNPs. Further functional analysis and search for correlations between genotype and phenotype will help to evaluate the role of these genes in the development of autoimmune thyroid disease. Susceptibility genes interact with thyroid autoimmunity [ 62 , 130 ], and the level of these interactions could affect disease severity and clinical expressions. The molecular mechanisms of these interactions is unknown. However, significant progress has been made in identifying susceptibility genes to HT and AITD along with intriguing findings regarding the functional characterization of disease-associated polymorphisms. These should stimulate further studies towards the in-depth understanding of the mechanisms by which these genes contribute to thyroid autoimmunity. Competing interests The author(s) declare that they have no competing interests. Authors' contributions ABFG | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555850.xml |
549074 | Insulin-like growth factor binding protein 2 promotes ovarian cancer cell invasion | Background Insulin-like growth factor binding protein 2 (IGFBP2) is overexpressed in ovarian malignant tissues and in the serum and cystic fluid of ovarian cancer patients, suggesting an important role of IGFBP2 in the biology of ovarian cancer. The purpose of this study was to assess the role of increased IGFBP2 in ovarian cancer cells. Results Using western blotting and tissue microarray analyses, we showed that IGFBP2 was frequently overexpressed in ovarian carcinomas compared with normal ovarian tissues. Furthermore, IGFBP2 was significantly overexpressed in invasive serous ovarian carcinomas compared with borderline serous ovarian tumors. To test whether increased IGFBP2 contributes to the highly invasive nature of ovarian cancer cells, we generated IGFBP2-overexpressing cells from an SKOV3 ovarian cancer cell line, which has a very low level of endogenous IGFBP2. A Matrigel invasion assay showed that these IGFBP2-overexpressing cells were more invasive than the control cells. We then designed small interference RNA (siRNA) molecules that attenuated IGFBP2 expression in PA-1 ovarian cancer cells, which have a high level of endogenous IGFBP2. The Matrigel invasion assay showed that the attenuation of IGFBP2 expression indeed decreased the invasiveness of PA-1 cells. Conclusions We therefore showed that IGFBP2 enhances the invasion capacity of ovarian cancer cells. Blockage of IGFBP2 may thus constitute a viable strategy for targeted cancer therapy. | Background Ovarian cancer is the most lethal gynecological malignancy. Indeed, epithelial ovarian cancer is detected at a late clinical stage in as much as 75% of patients, in whom the overall survival rate is a dismal 14–30% [ 1 ]. Hindering the development of effective treatments for the cancer is the fact that the molecular events responsible for the biological behavior of ovarian cancer are poorly understood. Implicated in both ovarian tumorigenesis and physiological follicular proliferation are the insulin-like growth factor I (IGF-I) and IGF-II systems. IGFs are regulated by at least six members of the IGF binding protein (IGFBP) family. High levels of IGFBP2 were detected in the serum or cystic fluid from patients with ovarian cancer compared with those with benign and borderline tumors [ 2 - 4 ]. A recent study further showed that IGFBP2 mRNA was overexpressed to a greater extent in advanced-stage serous ovarian cancer than normal ovarian tissue [ 5 ]. In addition, the increase in IGFBP2 expression was found to correlate positively with the levels of the serum tumor marker CA125 [ 4 ]. The progression-free interval and overall survival have also proved to be significantly shorter in patients with a high serum level of IGFBP2 at diagnosis than in those with lower levels [ 6 ]. Taken together, these data suggest an important role for IGFBP2 in the biology of ovarian cancer. IGFBP2 is also overexpressed in a wide spectrum of other cancers, including glioma, prostate cancer, synovial sarcoma, neuroblastoma, colon cancer, adrenocortical cancer, lung cancer, Wilms' tumor, and hepatoblastoma [ 7 - 17 ]. The overexpression of IGFBP2 also correlates with the aggressiveness of some tumors, including prostate cancer, hepatoblastoma and glioma [ 10 , 17 , 18 ], suggesting that IGFBP2 possesses a carcinogenic property. The observation that IGFBP2 has an RGD motif suggests that IGFBP2 modulates the integrin/cytoskeleton system. Indeed, IGFBP2 was recently found to interact with the alpha v beta 3 and alpha 5 beta 1 integrins [ 19 , 20 ]. In addition, IGFBP2 has been found to stimulate the growth of prostate cancer cells, an effect that can be blocked by MAP-kinase and PI3-kinase inhibitors [ 21 ]. IGFBP2 was also found to be co-expressed with the vascular endothelial growth factor in pseudopalisading glioma cells surrounding tumor necrosis [ 22 ]. Further, IGFBP2 enhances glioma cell invasion by increasing invasion-related genes including MMP2 [ 23 ]. These findings collectively suggest that IGFBP2 plays a key role in human cancer development. In this study, we found that overexpression of IGFBP2 enhanced the invasiveness of ovarian cancer cells. Further, attenuation of IGFBP2 expression by siRNA reduced the invasiveness of ovarian cancer cells. Results and Discussion IGFBP2 is associated with invasive epithelial ovarian carcinoma We first performed western blotting analysis using frozen tissue specimens, which consisted of four paired normal and carcinomatous ovarian tissues, one normal ovarian tissue, and three unpaired ovarian carcinoma tissues. This result showed that IGFBP2 was frequently overexpressed in ovarian cancer tissues compared with normal ovarian tissues (Figure 1A ). We then compared the expression of IGFBP2 in normal ovaries, borderline serous ovarian tumors, and invasive serous ovarian carcinomas using a tissue microarray, which showed that IGFBP2 was expressed at significantly different levels between normal tissues and borderline tumors ( p = 0.03) and between borderline tumors and invasive carcinomas ( p = 0.03) (Figure 1B , Table 1 ). Because borderline ovarian tumors have no obvious stromal invasion or infiltrative growth in contrast with invasive ovarian carcinoma [ 24 ], this finding suggests that IGFBP2 overexpression could induce invasion nature of ovarian cancer. Figure 1 Overexpression of IGFBP2 in ovarian carcinoma. (A) Western blotting analysis in paired normal and cancer tissues (N1 to T4), one normal ovarian (N5) and 3 unpaired ovarian cancer tissues (T6 to T8) showed frequent overexpression of IGFP2 in ovarian cancer tissues compared with normal ovarian tissues. (N: normal ovarian tissue, T: ovarian cancer tissue). (B) Expression of IGFBP2 in normal ovary (100×), borderline ovarian tumor (100×), and invasive ovarian carcinoma (200×) using tissue microarray. IGFBP2 was expressed at greater level in invasive ovarian carcinoma than normal and borderline ovarian tumors. Table 1 Increasing expression of IGFBP2 with progression of ovarian serous tumors*. Tissue IGFBP2 expression (Scores) P value 3 2 1 0 Normal Ovaries ◇ ◇◇◇◇◇ 0.03 # Borderline Ovarian Tumors ◇◇◇◇ ◇ ◇◇ ◇◇◇◇◇ ◇◇◇◇◇ Invasive Ovarian Carcinomas ◇◇ 0.03 § ◇◇◇◇◇ ◇◇◇◇◇ ◇◇◇◇ ◇◇◇◇ ◇◇◇◇◇ ◇◇◇◇◇ ◇◇◇◇◇ ◇◇◇◇◇ *Each diamond represents one case (Mann-Whitney U test). # P value between normal ovaries and borderline ovarian tumors § P value between borderline ovarian tumors and invasive ovarian carcinomas IGFBP2 enhances the invasiveness of ovarian cancer cells Thus far, the biological or pathophysiological role of IGFBP2 in ovarian cancer is unknown. To elucidate the role of increased IGFBP2 in ovarian cancer, we therefore generate IGFBP2-overexpressing cells. To obtain suitable cells for this study, we first examined the endogenous expression of IGFBP2 in six ovarian cancer cell lines: NIH:OVCAR3, SKOV3, PA-1, OV-90, TOV-112D, and TOV-21G. IGFBP2 was expressed at different levels in all six cell lines (Figure 2A ). Both SKOV3 and OV-90 cells showed very low levels of endogenous IGFBP2; NIH:OVCAR3, PA-1, and TOV-112D cells expressed IGFBP2 at high levels; and TOV-21G cells expressed IGFBP2 at a relatively moderate level. We thus selected SKOV3 cell line and generated two vector transfected clones and three IGFBP2-overexpressing clones by transfection (Figure 2B ). Figure 2 IGFBP2 overexpressing promotes ovarian cancer cell invasion. (A) IGFBP2 expression of six ovarian cancer cell lines. The western blotting analysis shows that the expression level of IGFBP2 is heterogeneous in cell lines. SKOV3 and OV-90 ovarian cancer cell lines have very low endogenous IGFBP2. NIH:OVCAR3, PA-1 and TOV-112D have high levels of IGFBP2 expression whereas TOV-21G has relatively moderate expression of IGFBP2. (B) Two vector transfected clones and three IGFBP2 stable clones with different expression level were obtained. The expression of IGFBP2 was determined by western blotting analysis (p: parental SKOV3 cell line, v: vector transfected cell lines, b: IGFBP2 stable cell lines). (C) The invasion capacity of stable clones showed that IGFBP2 overexpressing cells have invasion potential as 1.84 – 2.89 fold as parental and vector transfected cells. (* p < 0.05) We studied the cellular phenotype of IGFBP2-overexpressing cell lines. Similar to observations in gliomas [ 23 ], we did not observe differences in cell proliferation (data not shown). This is contrary to the findings in prostate cancer, adenocortical cancer and neuroblastoma, in which IGFBP2 has been found to stimulate cell proliferation [ 21 , 25 , 26 ]. We then assessed the invasive capacity by a Matrigel in vitro invasion assay. The invasiveness of the IGFBP2-overexpressing cells was 1.8- to 2.9-fold greater than that of the vector-alone-transfected cells ( p < 0.05; Figure 2C ). Our tissue microarray findings were consistent with this finding. This suggests that acquisition of IGFBP2 is a very important step in the penetration of the extracellular matrix by ovarian cancer cells, which they may need to do before they can move to adjacent tissue and the lymphovascular space. This provides a site for occult progression or the formation of recurrent ovarian cancers. Indeed, ovarian cancer frequently spreads through the lymphatic system [ 27 ]. In fact, in one study, 20% of patients with early-stage invasive ovarian carcinoma whose tumors appeared to be confined to the ovary (stage I disease) were found to have lymph node metastasis [ 28 ]. The mortality rate in such patients is higher than that in patients without lymph node metastasis [ 29 ]. Our study therefore also provides strong evidence that IGFBP2 could be a factor indicating a poor prognosis. Disruption of IGFBP2 inhibits ovarian cancer cell invasion The observation that IGFBP2 increased the invasive capacity of ovarian cancer cells and those of previous studies prompted us to determine whether IGFBP2 could serve as a target of therapy for ovarian cancer. We used PA-1 ovarian cancer cells for this experiment because they express a high level of endogenous IGFBP2 and show a relatively high invasive capacity compared with other ovarian cancer cell lines. We designed siRNAs for four different target sites of IGFBP2 mRNA because not all siRNAs were expected to effectively attenuate IGFBP2 expression. Western blotting analysis showed that the IGFBP2 level after siRNA-3 transfection was comparable to the levels after the transfection of Lamin A/C and negative control siRNA in OVCAR3 and PA-1 ovarian cancer cell lines, suggesting that the siRNA-3 was not effective (Figure 3A ). Considering that not all siRNA molecules work, this was not surprising. Therefore, we used siRNA-3-transfected cells as a negative control. siRNA-1 and siRNA-4 attenuated IGFBP2 expression in the PA-1 cells more than did either siRNA-2 or siRNA-3 (Figure 3B ). Likewise, in the invasion assay, the invasive capacity of the cells transfected with siRNA-1 or siRNA-4 was significantly decreased compared with that of the cells transfected with siRNA-2 or siRNA-3 ( p < 0.05; Figure 3C ). These findings therefore support to the notion that IGFBP2 is a viable target of therapy for ovarian cancer. Figure 3 Attenuation of IGFBP2 inhibits ovarian cancer cell invasion. (A) Western blotting analysis of IGFBP2 after transfection of siRNA in OVCAR3 and PA-1. 4 siRNAs inhibit the IGFBP2 with various levels. IGFBP2 levels of siRNA-3 transfected cells have similar to those of Lamin A/C and negative control transfected cells. (B) Four different siRNAs were transfected to PA-1 ovarian cancer cells which has high endogenous IGFBP2. Different inhibiting levels of IGFBP2 were determined by western blotting analysis. siRNA-1 and -4 were working better than siRNA-2 and -3. (C) The invasion activity after 72 hours of siRNA transfection was significantly decreased in siRNA-1 and -4 treated cells comparing with siRNA-2 and -3 treated cells (* p < 0.05). Conclusions In this study, we showed that IGFBP2 was significantly overexpressed in invasive ovarian carcinomas compared with borderline ovarian tumors as well as normal ovarian tissues and that IGFBP2 increases invasion capability of ovarian cancer cells. These results provide evidence that IGFBP2 could promote ovarian cancer progression by augmenting the invasion potential. Although further investigations of molecular mechanisms are required, our findings using siRNA study support IGFBP2 as a novel target for the treatment of ovarian cancer. Methods Ovarian tissues and cell lines Ovarian cancer and normal control tissues were obtained from The University of Texas M. D. Anderson Cancer Center tumor tissue bank with the approval of the Institutional Review Board. The ovarian cancer cell lines NIH:OVCAR3, SKOV3, OV-90, TOV-112D, TOV-21G, and PA-1 were purchased from the American Type Culture Collection (Manassas, VA). NIH:OVCAR3 cells were maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS); SKOV3 and PA-1 cell were maintained in McCoy's 5a medium and Dulbecco's modified Eagle medium (DMEM)/F12, respectively, supplemented with 10% FBS; OV-90, TOV-112D, and TOV-21G cells were maintained in a 1:1 mixture of MCDB 105 medium and medium 199, supplemented with 15% FBS. All cells were kept at 37°C in a humidified atmosphere with 5% CO 2 . Media were routinely changed every 3 days. Construction of progression tissue microarray for ovarian serous tumors Formalin-fixed, paraffin-embedded archival tissue blocks from ovarian cancer patients who had undergone surgery at The University of Texas M. D. Anderson Cancer Center between 1990 and 2001 were used to construct progression tissue microarrays according to previously described methods [ 30 ]. The progression tissue microarray consisted of normal ovarian surface epithelium from 6 individuals, serous borderline tumors from 17 patients, and invasive serous carcinomas from 40 patients. Tissue cores with a diameter of 1.0 mm were obtained from each sample and assembled into two separate tissue array blocks. Immunohistochemistry studies Polyclonal antibody against IGFBP2 (c-18; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used in the immunohistochemistry studies. This antibody is specific and does not cross-react with other isoforms of IGFBP. A standard indirect immunoperoxidase procedure (ABC-Elite; Vector Laboratories, Burlingame, CA) was used for all stains. In brief, antigen retrieval was performed by first placing unstained slides in a steamer for 25 min. The antibody against IGFBP2 (in a 1:1000 dilution) was overlaid on the tissue sections of tissue arrays, and incubation was performed at 4°C overnight. Secondary antibody incubation was performed at room temperature for 60 min. Mayer's hematoxylin nuclear stain was used as a counterstain. Staining intensity was graded on a 0–3 scale, where 0 = no staining as assessed by staining with anti-goat secondary antibody alone, 1 = weak (<10%), 2 = moderate (10–50%), and 3 = strong (50–100%). The results of the immunohistochemistry studies were statistically analyzed using a Mann-Whitney nonparametric U test. Stable clone establishment To establish stable cell lines that overexpressed IGFBP2, we transfected SKOV3 ovarian cancer cell lines with a pcDNA3.1 expression vector encoding IGFBP2 cDNA using FuGENE6 reagent (Roche Diagnostics Corporation, Indianapolis, IN). Transfected cells were subsequently selected in the presence of G418 (300 μg/ml) for 5 weeks. The expression of IGFBP2 clones was determined from western blots of cell extracts with anti-IGFBP2 antibody (C-18). Two vector-transfected cell lines and three IGFBP2 stable cell lines were used in this study. Established stable cells were maintained without antibiotics. Western blot analysis Equal amounts of proteins from the total cell lysates was separated by 10% SDS-PAGE and transferred electrophoretically to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Chicago, IL). The membrane was blocked in 5% skim milk in 1× PBS and probed with a 1:1000 dilution of a goat polyclonal anti human IGFBP2 (C-18) overnight at 4°C. An enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) was used to visualize the proteins. In vitro chemoinvasion assay We used 24-well BioCoat Matrigel invasion chambers (Becton Dickinson Labware, Bedford, MA) with an 8-μm pore polycarbonate filter coated with Matrigel to measure chemoinvasion. The lower compartment contained 0.75 ml of medium with 0.5% FBS as a chemoattractant. In the upper compartment, 5 × 10 4 to 2 × 10 5 cells/well were placed in triplicate wells and incubated for 22 h at 37°C in a humidified incubator with 5% CO 2 . After incubation, the cells that had passed through the filter into the lower wells were stained with Giemsa (Fisher Scientific, Orangeburg, NY) and counted by analyzing images under a microscope using software program as described previously [ 31 ]. All assays were repeated at least three times. Student's t-test was used to analyze the differences in the invasion rates between control cell lines and stable cell lines. A p value of <0.05 was considered statistically significant. siRNA transfection Four different siRNA molecules designed for IGFBP2 mRNA and siRNA molecules for Lamin A/C and negative control were synthesized and purified (Qiagen, Valencia, CA). siRNA that targets Lamin A/C and siRNA that bears no homology with relevant human genes was used as a negative control. AATGGCGATGACCACTCAGAA was the target sequence for siRNA1; AAGGGTGGCAAGCATCACCTT was the target sequence for siRNA2; AAGCGCCGGGACGCCGAGTAT was the target sequence for siRNA3; AACCTCAAACAGTGCAAGATG was the target sequence for siRNA4; AACTGGACTTCCAGAAGAACA was the target sequence for Lamin A/C; and AATTCTCCGAACGTGTCACGT was the target sequence for the negative control. siRNAs were dissolved in siRNA suspension buffer to a final concentration of 20 μM, and the mixture was heated to 90°C for 1 min and incubated at 37°C for 60 min. PA-1 ovarian cancer cells (2 × 10 5 ) were plated to a 6-well plate and allowed to adhere for 24 h; the confluency of the cell monolayer at the time of transfection was 40–60%. 5 μg of siRNA and 15 μl of RNAiFect Transfection Reagent (Qiagen, Valencia, CA) was used. The cells were incubated under normal cell culture conditions. All assays were performed 72 h after treatment. Authors' contributions EL carried out Western blotting analysis, establishing stable cells, invasion assay and siRNA transfection, participated in the analysis of all data and drafted the manuscript. CM and IS contributed to the statistical analysis. HW and JL provided frozen tissues and carried out tissue microarray and its analysis. AN performed the analysis of invasive assay. JK participated in its design of the study. JL and WZ participated in its design and coordination and helped draft the manuscript. All authors read and approved of the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549074.xml |
552332 | Platelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways | Background A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM. Results Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-β1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-β1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-β1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did. Conclusion These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis. | Background Angioplasty is a procedure designed to treat vascular stenosis, blockage(s), or atherosclerotic lesions, but it may also, simultaneously, cause damage to the integrity of the blood vessel wall. Restenosis is the subsequent narrowing and occlusion of the blood vessel in response to the injury or damage sustained during angioplastic procedures such as balloon dilation [ 1 ]. During restenosis, vascular smooth muscle cells (VSMC) from the injured blood vessel wall migrate into the lumen of the vessel, creating a new or neointima. The subsequent proliferation of these neointimal VSMC can lead to a thickening of this neointimal layer and re-occlusion of the vessel [ 1 ]. The characteristic response of VSMC, endothelial cells, platelets, and macrophages at the site of injury is the release of specific soluble growth factors which include platelet-derived growth factor (PDGF), transforming growth factor (TGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) [ 2 - 4 ]. VSMC of the vessel wall respond to these factors by secreting proteolytic matrix metalloproteinases that degrade the extracellular matrix (ECM) and stimulate deposition of new ECM proteins such as collagen, elastin, fibronectin, and laminin in the neointima [ 5 - 7 ]. These ECM modulate VSMC integrin-dependent behaviors such as transluminal migration, adhesion, and proliferation [ 8 - 10 ]. To date, the precise molecular mechanisms that link growth factor-initiated intracellular signaling to ECM-mediated adhesion, migration, and proliferation of VSMC are still unknown. Our previous reports of low-level laminin-5 expression in the intima of normal vasculature and an increased expression of laminin-5 in the neointima of injured vessels suggest that laminin-5, in addition to PDGF and TGF, may mediate VSMC responsiveness following vascular injury [ 11 - 13 ]. To further elucidate VSMC response to growth factors and the intracellular signaling cascades that may be linked to ECM-mediated adhesion, we used in vitro assays to study the role of laminin-5 in modulating these behaviors in rat vascular smooth muscle cells (rVSMC). We report here that PDGF induces differential responses in rVSMC behaviors on laminin-5, but not on fibronectin. In addition, we find that the PDGF-induced responses on laminin-5 are inhibited in a dose-dependent manner by an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD98059, but not on fibronectin. These differences in MAPK-sensitive rVSMC responses in vitro may be the result of different signaling pathways that are initiated by integrin-mediated adhesion to laminin-5. We suggest that rVSMC binding to laminin-5 may initially activate a MAPK-independent signaling cascade that may make these cells more responsive to MAPK inhibition. In contrast, rVSMC binding to fibronectin may activate a signaling pathway shared by PDGF that ultimately converges in MAPK activation. These results suggest that a complex interaction of ECM and growth factors may closely regulate VSMC behavior following vascular injury and these studies may directly identify define molecular targets that may reduce the incidence of restenosis following angioplasty. Results Migration We have previously reported that laminin-5 expression by rat vascular smooth muscle cells (rVSMC) is upregulated by platelet-derived growth factor (PDGF-BB) and that laminin-5 specifically enhances PDGF-BB-stimulated rVSMC migration [ 12 ]. In addition, our previous results suggested that PD98059 (a specific inhibitor of MEK and a general inhibitor of the mitogen-activated protein kinase ERK1/2 pathway) blocked PDGF-BB-stimulated migration on laminin-5 [ 13 ]. The current study characterizes this synergistic relationship between PDGF-BB and laminin-5 in the modulation of rVSMC cell behaviors, including migration. The level of rVSMC migration was observed over 18 hours in transwell migration filters in the presence or absence of laminin-5, with and without PDGF-BB or serum. Maximal cell migration (chemotaxis) was obtained using cell migration media containing ten (10) percent fetal calf serum (FCS). Similar to our previous reports, laminin-5-coated wells induced a greater than four-fold increase in rVSMC migration over that measured in naked plastic controls in three independent experiments as shown in Figure 1 (n = 24, p < 0.05). Furthermore, PDGF-BB, tested over a biologically relevant range of 5 – 50 ng/mL, stimulated a dose-dependent increase in rVSMC migration on laminin-5 (between 5 – 25 ng/mL), increasing migration fifteen (15) to sixty (60) percent over laminin-5-stimulated migration alone (n = 24, p < 0.005). This PDGF-BB-stimulated increase in migration peaked at 25 ng/mL and did not significantly increase over the range of 25 – 50 ng/mL (data not shown). Figure 1 PDGF-BB increased rVSMC migration on laminin-5 in vitro . The addition of PDGF-BB increased rVSMC migration on laminin-5, in a dose-dependent manner, over rVSMC haptotactic migration induced by the presence of laminin-5. rVSMC migration on laminin-5 was inhibited by both PD98059 (MEK1 inhibitor) and TGF-β1, in a dose-dependent manner. The mitogen-activated protein kinase (MAPK) pathway is known to be activated by PDGF-BB-stimulation in VSMC [ 14 ]. To explore the mechanism of increased rVSMC migration on laminin-5 via PDGF-BB-stimulation, we used PD98059 which is a specific inhibitor of MEK1 and MEK2, and a general inhibitor of MAPK activation. The addition of PD98059 in concentrations between 10 – 50 ng/mL reduced PDGF-BB-stimulated migration on laminin-5 (25 ng/mL) in a dose-dependent manner, reaching a maximal reduction of sixty (60) to seventy (70) percent over the range of 20 – 40 ng/mL, as shown in Figure 1 (n = 24, p < 0.005). This reduction in PDGF-BB-stimulated migration on laminin-5 was maintained at all concentrations of PDGF-BB tested (5 – 25 ng/mL). To determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1) for its ability to modulate the PDGF-stimulated increase in rVSMC migration on laminin-5. Our results indicated that the addition of TGF-β1 was able to reduce laminin-5-stimulated rVSMC migration to levels approximating the levels observed in naked plastic controls over all tested ranges (5 – 50 ng/mL), although the most statistically distinct reduction was found over the range of 5 – 25 ng/mL, as shown in Figure 1 . In addition, TGF-β1 was able to reduce maximal PDGF-BB-stimulated migration of rVSMC (25 ng/mL) on laminin-5 by approximately fifty (50) to sixty (60) to percent, over the range of concentrations from 20 to 40 ng/mL. Adhesion To determine whether or not the PDGF-BB-stimulated increase in rVSMC migration correlates with a reduction in rVSMC adhesion to laminin-5, thirty-minute in vitro adhesion assays were performed. In the absence of exogenous growth factor stimulation, laminin-5- and fibronectin-coated wells (20 μg/mL) sustained an approximate two and a half-fold increase in rVSMC adhesion compared with negative controls, as shown in Figure 2 (n = 24, p < 0.05). The addition of PDGF-BB, at the maximal migration-stimulating dose of 25 ng/mL, decreased the adhesion of rVSMC on laminin-5 by more than sixty-five (65) percent (n = 24, p < 0.005). The addition of exogenous PDGF-BB (25 ng/mL), however, decreased rVSMC adhesion on fibronectin by less than thirty (30) percent. Figure 2 PDGF-BB reduces rVSMC adhesion on laminin-5 adhesion in vitro . The presence of laminin-5 supported greater adhesion of rVSMC over naked plastic, and this increase in adhesion was reduced by the addition of PDGF-BB. Inhibition of MEK1, using PD98059 restored rVSMC adhesion to laminin-5 in the presence of PDGF-BB. Although fibronectin also supported rVSMC adhesion, the effect of adding PDGF-BB was less pronounced and was not restored using PD98059. The addition of TGF-β1, however, completely restored rVSMC adhesion to both fibronectin and laminin-5 in the presence of PDGF-BB. To investigate if the relationship between the PDGF-BB-stimulated increase in migration and corresponding decrease in adhesion of rVSMC on laminin-5 may be related to MAPK activation, cells were pre-treated with PD98059 (40 ng/mL) for twenty (20) minutes prior to assay and the adhesion media was supplemented with PD98059 (40 ng/mL). The addition of exogenous PD98059 (40 ng/mL) restored the PDGF-BB-stimulated reduction (25 ng/mL PDGF-BB) in rVSMC adhesion to laminin-5, to approximately eighty-five (85) percent of laminin-5 controls (n = 24, p < 0.05). The addition of PD98059, however, did not restore the thirty (30) percent PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin. To determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1). Our results indicated that the addition of TGF-β1 (25 ng/mL) was able to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on laminin-5 by roughly the same level as PD98059, eighty two (82) percent versus eighty five (85) percent, respectively. In contrast to PD98059, the addition of TGF-β1 (25 ng/mL) was sufficient to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin. Proliferation Based upon our observations of rVSMC migration and adhesion, we performed in vitro proliferation assays to determine the relative effects of the extracellular matrix (ECM) and exogenous growth factors described above. First, to test the effects of the ECM substrate on rVSMC proliferation in the absence of exogenous growth factors, quiescent cells were plated in cell-culture plates either coated with or without laminin-5 or fibronectin, in serum-free Dulbecco's Modified Eagle's Medium (DMEM) for one (1) to six (6) days. To induce quiescence, rVSMC were incubated for forty eight (48) hours without mitogen (0% FCS, FBS) at 37°C and quiescence was verified by proliferation controls, cultured for forty eight (48) hours at 37°C with mitogen stimulus as shown in Figure 3a and 3b . Previous studies with VSMC report that greater than 95% of cells incubated in low-serum media (0.4% FCS or less) were arrested in G 0 (G 1 ) between forty eight (48) and seventy two (72) hours as determined by flow cytometry and determination of [3H] thymidine-labeled nuclei [ 15 ]. Figure 3 The effect of growth factors on rVSMC proliferation in vitro . The addition of PDGF-BB stimulated proliferation of rVSMC on laminin-5 and to some extent on naked plastic, but not on fibronectin. The presence of fibronectin alone was able to stimulate proliferation of rVSMC. The proliferative response of rVSMC to presence of laminin-5 with PDGF-BB or to fibronectin was suppressed by the addition of TGF-β1. Figure 3b The ECM induces differential rVSMC proliferation responses in vitro . The plating of rVSMC on fibronectin, but not laminin, induced increases in proliferation over four days. The addition of PDGF-BB, however, increased rVSMC migration on laminin-5, but not on fibronectin. The addition of TGF-β1 was sufficient to suppress rVSMC proliferation on both laminin-5 and fibronectin. Our results suggest that culture of rVSMC plated on exogenous laminin-5 (coated at a concentration of 20 μg/mL) did not significantly increase cellular proliferation compared with naked plastic controls over a period of four (4) days, as shown in Figure 3a (n = 24, p < 0.02). Culturing of rVSMC plated on exogenous fibronectin (coated at a concentration of 20 μg/mL) however, did significantly increase cellular proliferation over a period of four (4) days by nearly two-fold, as shown in Figures 3a and 3b . Next, to test the modulating effects of exogenous growth factors on rVSMC proliferation when cultured on these ECM substrates, quiescent cells were plated in cell-culture plates either naked or coated with laminin-5 or fibronectin, in the presence of TGF-β1 or PDGF-BB, both at a concentration of 25 ng/mL, from one (1) to six (6) days. Our results indicated that the addition of exogenous TGF-β1 (25 ng/mL) to the cell culture medium was sufficient to induce a suppressing effect on rVSMC proliferation on naked plastic, as well as laminin-5 and fibronectin, to levels approximating the quiescent growth controls. The addition of PDGF-BB was sufficient to induce an increase in rVSMC proliferation on laminin-5 of nearly one-half (46%) over laminin-5 DMEM (no serum, no mitogen) controls (n = 24, p < 0.01). However, our results indicate that the addition of exogenous PDGF-BB (25 ng/mL) did not have a statistically significant effect on cells plated on fibronectin. Mitogen Activated Protein Kinase (MAPK) Western Blot To examine MAPK activation, rVSMC were plated onto laminin-5- or fibronectin-coated plates, then lysed after thirty minutes and prepared for immunoblotting. Our results indicated that laminin-5 did not induce a detectable increase in MAPK activation over a thirty (30) minute time interval, as shown in Figure 4 . rVSMC plated on fibronectin did exhibit a significant increase in MAPK levels after thirty (30) minutes. Figure 4 The MAPK pathway in rVSMC is activated by different stimuli . The addition of FCS or PDGF-BB or the plating of rVSMC on fibronectin was sufficient to induce measurable increases in p44/p42 activation over 30 minutes. However, the addition of TGF-β1 or the plating of rVSMC on laminin-5 was not sufficient to induce MAPK activation. The addition of growth factors, such as PDGF-BB and TGF-β1, have been associated with increases in p44/p42 (ERK1/2) or MAPK activation [ 16 - 18 ]. To determine the effects of PDGF-BB and TGF-β1 on rVSMC, subconfluent cell cultures were pre-treated with FCS (10%), PDGF-BB (25 ng/mL) or TGF-β1 (25 ng/mL) for thirty (30) minutes, then lysed and prepared for immunoblotting. p44/p42 phosphorylation levels were not detectable in DMEM-treated control cells, as shown in Figure 4 . However, the addition of PDGF-BB, but not TGF-β1, was sufficient to induce measurable increases in MAPK activation levels. Discussion PDGF-BB is an in vitro VSMC mitogen and may be responsible for initiating the phenotypic changes in VSMC migration and proliferation during restenosis in vivo [ 19 , 20 ]. Our recent reports of low-level laminin-5 expression in the intima of normal arteries and increased expression in the neointima of injured arteries suggest that laminin-5, in conjunction with soluble growth factors and mitogens, may determine VSMC phenotype during restenosis [ 11 - 13 ]. The current study augments the body of evidence that suggests VSMC growth is influenced by an ECM-VSMC interaction [ 21 ] and that VSMC proliferation in different species respond differently to growth factor stimulus [ 15 ]. More specifically, this study explores evidence that laminin-5 and PDGF-BB may have combined and synergistic effects in determining rVSMC phenotype in vitro [ 6 , 8 , 10 ]. More specifically, our studies suggest that PDGF-BB strongly influences rVSMC behaviors such as migration. In addition, PDGF-BB significantly alters other cellular behaviors such as adhesion and proliferation on laminin-5, but only to a lesser extent on fibronectin. Because PDGF-BB stimulation increases the overall levels of intracellular MAPK, as well as MAPK phosphorylation, we sought to explore this signaling cascade to determine its role in modulating these rVSMC behaviors on laminin-5 [ 22 ]. Specifically, the ERK1/2 form of MAPK mediates signaling by PDGF-AA, PDGF-AB, and PDGF-BB in VSMC, as well as signaling through laminin-5 binding integrins, and is therefore the most likely signaling molecule to modulate these cellular behaviors [ 23 - 25 ]. Our results suggest that rVSMC behaviors in vitro , driven by PDGF-BB responsiveness, can be blocked by MAPK inhibition (via MEK1 inhibitor: PD98059) on laminin-5, but not on fibronectin. An additional signaling regulator, TGF-β1, has been implicated in the negative regulation and decreased rate of proliferation of VSMC stimulated with serum or PDGF [ 26 - 28 ]. Our results from this study indicate that TGF-β1, unlike the MEK1-inhibitor PD98059, was sufficient to block rVSMC behaviors on both laminin-5 and fibronectin. Specifically, the addition of TGF-β1 was able to reduce PDGF-BB-stimulated migration and proliferation of rVSMC on both ECM substrates and was also able to restore PDGF-BB-stimulated reductions in VSMC adhesion on these ECM. Several lines of evidence now suggest that the anti-mitogenic effects of TGF-β1 may be dissociated from inhibition of ERK1/2 signaling pathways [ 17 , 18 ]. These reports suggest that TGF-β1 inhibition of PDGF-BB may be temporally independent of other early signaling pathways, such as MAPK, and is more likely to block VSMC behaviors, such as proliferation, by inhibiting events later in the G 1 phase of mitosis. Although our previous reports linked ERK1/2 to rVSMC adhesion and migration, these studies did not examine the possibility for differential signaling initiated by rVSMC binding to laminin-5 or fibronectin [ 12 , 13 ]. Expanding our original analysis of growth factor stimulation of MAPK to include ECM binding reveals that integrin binding of rVSMC to fibronectin strongly increases detectable MAPK activation levels, as does FCS and PDGF-BB stimulation, whereas binding to laminin-5 does not. These differences may help to explain the differing effects on cellular behaviors of binding to these ECM ligands, as fibronectin and PDGF-BB may act in unison to activate intracellular signaling cascades that converge in MAPK activation, while laminin-5 may not. Conclusions Our results indicate that laminin-5 activates different intracellular signaling pathways from those of fibronectin and PDGF-BB in rVSMC and that binding to laminin-5 may modulate rVSMC behaviors that are distinctive from those modulated by fibronectin. Although binding of rVSMC to laminin-5 may not cause an initial increase in MAPK activation levels or proliferation, laminin-5 can augment PDGF-BB-stimulated proliferation and migration of rVSMC in vitro . Based upon these findings, we postulate that PDGF-BB and laminin-5 binding may initially activate different intracellular signaling cascades, causing rVSMC to be more responsive to the inhibition of MEK1 and MAPK on laminin-5 than those activated on fibronectin, as outlined in Figure 5 . Figure 5 Integrins and growth factors activate intracellular signaling cascades in rVSMC. Intracellular signaling pathways that converge through MEK and ERK activation, may be initiated in rVSMC by the addition of PDGF-BB, as well as binding to laminin-5 or fibronectin. The MEK1 inhibitor PD98059 blocks MEK1 activation and alters rVSMC responses to PDGF-BB on laminin-5, but not fibronectin. The addition of TGF-β1, which may block later events in the cell cycle, is sufficient to block PDGF-BB induced responses of rVSMC on both laminin-5 and fibronectin. In contrast to laminin-5, fibonectin and PDGF-BB may have parallel, reinforcing roles in MAPK activation. Our analysis of the effects of TGF-β1 demonstrated that TGF-β1 does not strongly activate MAPK in rVSMC, but rather strongly inhibits the effects of fibronectin and PDGF-BB-stimulation on laminin-5. Our results support the previous findings that TGF-β1 may inhibit mitogenesis and other VSMC behaviors via mechanisms independent of MAPK activation. These results suggest that a clear understanding of the roles and contributions of each ECM may provide new insights into the mechanisms of regulating rVSMC cell behaviors, including migration, adhesion, and proliferation. Further analysis of the events that trigger and sustain the underlying cellular mechanisms of rVSMC behaviors may help aid in the design of more effective therapies for the treatment of restenosis. Methods Cell culture Cells were maintained in 100 mm × 20 mm Corning tissue-culture dishes (Plainfield, NJ) at 37°C and 5% CO 2 in humidified chambers. Cells were maintained in DMEM High Glucose, supplemented with 10% fetal bovine serum and 1% L-glutamine (29.2 mg/mL), penicillin G (10,000 U/mL), and streptomycin sulfate (10,000 mcg/mL) (GPS) from Irvine Scientific (Santa Ana, CA). Rat aortic smooth muscle cell explants were a gift from RC Smith and were isolated and passaged as previously described [ 29 ]. Although greater than 95% quiescence, G 0 (G 1 ) arrest can routinely be induced by incubation of cells for 72 h. in low-mitogen (0.5% FBS) medium [ 15 , 30 ], these authors suggest that incubation of VSMC for 48 h. without mitogen (0% FBS) is sufficient to induce quiescence. This was verified by proliferation control cells, cultured for 48 h. at 37°C with and without mitogen stimulus. Migration assays Cell migration assays were performed in Costar transwell filter plates either coated with purified matrix (laminin-5 or fibronectin) at a protein concentration of 20 μg/mL for one hour (60 min.) at room temperature, 25°C, and washed twice with phosphate-buffered saline 0.2% Tween-20 and 5% skim milk (PBST) prior to assay as previously described [ 31 , 32 ]. Cells were seeded at a concentration of 1.2 × 10 5 in each of 96-transwell chamber filters (100 μL of 1.2 × 10 6 cells/mL solution) with and without ECM in the presence or absence of PDGF-BB at the indicated concentrations (5–25 ng/mL) and allowed to migrate for 18 hours at 37°C. Where applicable, the medium was supplemented with PD98059 (MEK1-inhibitor) at the indicated concentration. Cells were counted at the end of an 18-hour interval as indicated, quantified with the following modification. 30 minutes prior to measuring migration, 5 μM calcein AM from Molecular Probes (Eugene, OR) was added to the migration wells at 37°C. To quantitate migration, cells were removed from the top of the filter with cotton-tipped applicators and fluorescence of the incorporated calcein was measured from the bottom of the filter with a fluorescence plate reader. Relative fluorescence values for each experimental condition are expressed relative to controls and untreated samples. Adhesion assays Cell adhesion assays were performed as previously described [ 31 , 32 ] using Costar 96-well cell culture cluster plates, coated with either laminin-5 or fibronectin solution at a protein concentration of 20 μg/mL for 1 hour (60 min.) at room temperature, 25°C. Wells were then washed twice with PBST prior to assay. Cells were seeded at a concentration of 1.2 × 10 5 in each of 96-transwell chamber filters (100 μL of 1.2 × 10 6 cells/mL solution) with and without ECM-coating (described above) in the presence or absence of PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or both, and allowed to attach for 30 minutes at 37°C. Where applicable, cells were first incubated for 20 minutes with PD98059 (40 ng/mL), a MEK1 inhibitor from New England Biolabs (Beverly, MA) at 37°C and the adhesion assay culture medium was supplemented with PD98059 at 40 ng/mL. Following adhesion, non-adherent cells were removed by suspending plates upside down in a rotating tank of PBS for 10 minutes at room temperature, 25°C. Adherent cells were then fixed and stained and the relative absorbance was measured using a TECAN-SPECTRAFluor spectrophotometer (TECAN, Durham, NC) at 595 nm. Proliferation assays Tissue culture plates were coated with purified fibronectin from Calbiochem (La Jolla, CA) or laminin-5 from Demos (La Jolla, CA) at a 20 μg/mL protein concentration for 1 hour (60 min.) at room temperature, 25°C as previously described [ 31 ]. Cells were seeded at a concentration of 1.2 × 10 5 in 100 mm 2 cell culture plates with and without fibronectin or laminin-5 and allowed to attach overnight (12 h.) at 37°C. Cells were then starved in serum-free DMEM for 48 hours to induce quiescence at 37°C, as outlined in Cell Culture Methods below. The medium was then replaced with fresh medium containing 25 ng/mL of TGF-β1 or PDGF-BB obtained from Calbiochem (La Jolla, CA) and incubated at 37°C. Cells were removed from culture wells with trypsin/EDTA and counted using trypan blue stain from Gibco Life Technologies (Rockville, MD) and a VWR Scientific Counting Chamber (Plainfield, NJ) at 24 hour intervals, from 1 – 6 days. Western Blot analysis Quiescent VSMC were pre-treated with culture medium containing 10% FCS, PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or plated on laminin-5- or fibronectin-coated tissue culture plates with DMEM for 30 minutes at 37°C. Subconfluent cell cultures were then lysed using ice-cold, 1X lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, pH 7.5) and boiled in SDS Boiling Buffer prior to analysis as previously described [ 33 ]. Proteins were separated by 7.5% SDS-PAGE and transferred to Immobilon-P transfer membranes from Millipore (Bedford, MA), incubated with primary antibody (1:200 dilution at 4°C overnight, 12 h.), secondary antibody (1:2000 dilution at 25°C for 1 h.), and then exposed to NEN CDP-Star chemiluminescence reagent (5–7 min.) and developed on Kodak X-OMAT LS scientific imaging film for subsequent analysis. Antibodies used for this analysis were anti-phospho ERK (p42/p44) rabbit polyclonal IgG primary antibody and goat anti-rabbit IgG-AP secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Statistics The differences between untreated and treated cell populations were measured using a t distribution. All samples were measured using two-tailed t tests as departure from normality can make more of a difference in a one-tailed than in a two-tailed t test. So long as the sample size is even moderate (>20) for each group, quite severe departures from normality make little practical difference in the conclusions reached from these analyses [ 34 ]. Competing Interests The author(s) declare that they have no competing interests. Authors' contributions KK carried out the migration, adhesion, and proliferation assays, the Western Blot analysis and assisted with experimental design. GEP conceived, monitored, and coordinated the experimental design. Both KK and GEP contributed equally to the writing of this manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552332.xml |
549048 | Are ICD-10 codes appropriate for performance assessment in asthma and COPD in general practice? Results of a cross sectional observational study | Background The increasing prevalence and impact of obstructive lung diseases and new insights, reflected in clinical guidelines, have led to concerns about the diagnosis and therapy of asthma and COPD in primary care. In Germany diagnoses written in medical records are used for reimbursement, which may influence physicians' documentation behaviour. For that reason it is unclear to what respect ICD-10 codes reflect the real problems of the patients in general practice. The aim of this study was to assess the appropriateness of the recorded diagnoses and to determine what diagnostic information is used to guide medical treatment. Methods All patients with lower airway symptoms (n = 857) who had attended six general practices between January and June 2003 were included into this cross sectional observational study. Patients were selected from the computerised medical record systems, focusing on ICD-10-codes concerning lower airway diseases (J20-J22, J40-J47, J98 and R05). The performed diagnostic procedures and actual medication for each identified patient were extracted manually. Then we examined the associations between recorded diagnoses, diagnostic procedures and prescribed treatment for asthma and COPD in general practice. Results Spirometry was used in 30% of the patients with a recorded diagnosis of asthma and in 58% of the patients with a recorded diagnosis of COPD. Logistic regression analysis showed an improved use of spirometry when inhaled corticosteroids were prescribed for asthma (OR = 5.2; CI 2.9–9.2) or COPD (OR = 4.7; CI 2.0–10.6). Spirometry was also used more often when sympathomimetics were prescribed (asthma: OR = 2.3; CI 1.2–4.2; COPD: OR = 4.1; CI 1.8–9.4). Conclusions This study revealed that spirometry was used more often when corticosteroids or sympathomimetics were prescribed. The findings suggest that treatment was based on diagnostic test results rather than on recorded diagnoses. The documented ICD-10 codes may not always reflect the real status of the patients. Thus medical care for asthma and COPD in general practice may be better than initially found on the basis of recorded diagnoses, although further improvement of practice patterns in asthma and COPD is still necessary. | Background There is broad agreement between existing guidelines for asthma [ 1 - 4 ] regarding diagnostic procedures, patient education and medical treatment [ 5 ]. The statements for chronic obstructive pulmonary disease (COPD) [ 6 - 8 ] are also consistent, despite the lack of evidence for some recommendations [ 9 ]. However, it has been shown previously that the management of these diseases in practice was not fully consistent with the guidelines [ 10 - 12 ], which may be related to the variety of views on the treatment of these conditions [ 13 , 14 ]. Furthermore, the knowledge about the diseases is changing rapidly and the management has become more complex [ 15 , 16 ]. Many patients with asthma or COPD are managed in primary care and it is important to optimise the treatment given the prevalence and impact [ 17 ]. However, it remains difficult to draw a clear picture of management of obstructive lung diseases in primary care since the diagnoses of asthma and COPD often have not been separated in many surveys [ 7 ]. This could be due to some diagnostic and therapeutic overlap between these both diseases. Especially in primary care it is difficult to distinguish both entities when the symptoms are presented in an early stage [ 18 ]. In particular the treatment response in this stage often seem to be not strongly related to the stated diagnoses [ 19 , 20 ], and also the treatment response of COPD in the long term course is disappointing [ 21 ]. Furthermore, systematic registration of diseases in primary care are often hampered by missing of recorded diagnoses [ 22 ] or discrepancies between diagnoses and prescribed medications [ 23 ]. A specific problem for health services research and performance assessment in Germany might be that diagnoses saved in medical records are used for reimbursement. For that reason it is unclear to what respect ICD-10 codes reflect the real problems of patients in general practice. It may be possible that physicians enter diagnoses in their records to get reimbursement, but use other information such as results of diagnostic tests for treatment. Empirical evidence on this phenomenon is limited, but its existence would complicate the assessment of practice patterns. The aim of this study was to assess the appropriateness of the recorded diagnoses and to determine what diagnostic information is used to guide medical treatment. For this purpose we analysed associations between recorded diagnoses, diagnostic procedures and medical treatment regarding asthma and COPD in general practice. Methods Study design A cross-sectional observational study was performed in six computerised general practices with a total of eleven GPs in the Rhine-Neckar region in Southern Germany. Setting and study subjects All patients who had attended their physicians with asthma, COPD or other lower airway diseases from January until June 2003 were included in the study. Ethical approval The study was approved by the Medical Ethics Committee of the University of Heidelberg. Procedure for selection of subjects Patients were identified by searching the electronic files for documented diagnoses concerning lower airway diseases within the time frame. The data were evaluated systematically according to the international classification of diseases (ICD-10). In detail, the practice software was checked for the ICD-10-codes of the chapter "chronic lower airway disease": J40 (bronchitis, not specified as acute or chronic), J41 (simple and mucopurulent chronic bronchitis), J42 (unspecified chronic bronchitis), J43 (emphysema), J44 (chronic obstructive pulmonary disease), J45 (asthma), J46 (status asthmaticus), J47 (bronchiectasis). Additionally, the ICD-10-codes J98 (diseases of bronchus, not elsewhere classified) and R05 (cough / bronchial hyperreactivity), and the repeated documentation of ICD-10-codes belonging to the chapter "acute lower airway disease" were extracted in order to detect potentially existing but not detected asthma or COPD. The chapter "acute lower airway disease" includes the diagnoses J20 (acute bronchitis), J21 (acute bronchiolitis) and J22 (unspecified acute lower respiratory infection). The performed diagnostic procedures and the actual medication for each identified patient were extracted and documented manually by two independent scientific fellows (L.G., I.M.) of the department. The variables of the used protocol for documentation are listed in table 2 . Table 1 Baseline characteristics Overall (n = 857) Asthma (n = 255) COPD (n = 112) Asthma + COPD (n = 25) Other (n = 465) age 41.84 ± 22.18 (min 1; max 97) 36.48 + 21.00 (min 1; max 88) 56.28 + 18.38 (min 21; max 92) 46.23 + 19.16 (min 26; max 71) 41.06 + 22.32 (min 1; max 97) sex m 385; f 472 m 121; f 134 m 55; f 57 m 15; f 10 m 194; f 271 Analysis Every ICD10-code specified above was extracted and written down, at maximum up to three ICD-10-Codes per patient were found. The diagnoses were clustered into three groups: asthma, COPD, and other lower airway diseases. If more than one diagnosis was documented, asthma (J45) or COPD (J44) were extracted as the leading diagnosis. The ICD-10 group J43 (emphysema) was included into the ICD-10 group J 44 (COPD). The ICD-10 groups other than J45 (asthma), J44 or J43 were merged into the group "other lower airway diseases". The performed diagnostic procedures were extracted manually. Four ways of making diagnoses have been found in the records: 1. taking medical history only, 2. taking medical history plus trial of medication, 3. taking medical history and performing spirometry, 4. taking medical history plus single measurement of PEF. The additional performance of bronchial challenge testing and chest X-ray was documented, too. Baseline data were evaluated descriptively. The t-test was used to detect differences of age. Linear logistic regression was used to test relationships between diagnosing asthma or COPD and the performed diagnostics. The relation between prescribing patterns and performed diagnostics was also examined with logistic regression analysis. Results Overall, 8765 patients attended the practices within the time frame. 857 (9,8%) patients had lower airways diseases (385 male, 472 female). A total of 255 (2.9% of 8765) had the recorded diagnosis of asthma and 112 (1.3% of 8765) had COPD (107 with J44 = COPD, 5 with J43 = emphysema). A combination of asthma and COPD was documented in 25 (0.2% of 8765) cases. The 465 (5.3% of 8765) patients with "other lower airway diseases" included seven cases with acute bronchitis (J20), none with acute bronchiolitis (J21), one case with unspecified acute lower respiratory infection (J22), 251 (2.9% of 8765) cases with not specified bronchitis (J40), 38 with simple and mucopurulent chronic bronchitis (J41), 57 with unspecific chronic bronchitis (J42), one with bronchiectasis (J47), seven with diseases of bronchus, not classified (J98), 103 (1.2% of 8765) with cough / bronchial hyperreactivity (R05), Patients with COPD were significantly older than those with asthma or other lower airway diseases. There was no case of twofold documentation of "acute lower airway disease" (J20 - J22) within the time frame. The details regarding the patient population are described in table 1 . Table 2 Performed diagnostics and documented diagnoses Asthma (n = 255) COPD (n = 112) Other (n = 465) Performed diagnostics n % OR 95% CI n % OR 95% CI n % OR 95% CI Medical history only 42 16.5 0.11 0.07–0.15 33 29.3 0.31 0.20–0.46 338 72.7 10.9 7.89–15.02 Medical history and trial of medication 57 22.4 8.11 4.68–14.08 6 5.4 0.51 0.23–1.13 12 2.6 0.14 0.07–0.26 Medical history and spirometry 77 30.2 1.30 0.99–1.72 65 58.1 4.38 2.99–6.40 90 19.4 0.37 0.27–0.50 Medical history and PEF-measurement 79 31.0 7.57 4.93–11.60 8 7.1 0.81 0.47–1.40 25 5.4 0.18 0.11–0.28 Additional bronchial challenge test 18 3.9 1.35 0.73–2.48 24 21.4 6.74 3.66–12.42 1 0 0.16 0.08–0.35 Additional chest X-ray 34 13.3 0.88 0.58–1.31 42 37.5 4.06 2.67–6.19 48 10.3 0.44 0.30–0.65 Diagnostics Table 2 shows the performed diagnostic procedures. Spirometry was performed in 30% of the patients with recorded asthma. A high amount was diagnosed with 'medical history and single measurement of peak expiratory flow' (31%), and 22% of cases fell into the category of medical history taking plus trial of medication (= diagnosing by therapy). This implies that the physician gives sympathomimetics in suspected asthma. If the patient feels better, the diagnosis 'asthma' is made. In about 17% the diagnosis asthma was made only on the basis of medical history Spirometry was performed in 58% of the cases with recorded COPD. In 29% the diagnosis was made only by medical history. A small percentage (7%) was diagnosed by history taking and PEF-measurement and 5% by diagnosing by therapy. Concerning the other lower airway diseases, spirometry was done in 19% in order to exclude asthma or COPD. These diagnoses were made mainly on the basis of medical history (73%). The associations between recorded diagnoses and diagnostic procedures are calculated by binary logistic regression (also in table 2 ). The higher the odds ratio (OR) the higher is the positive association between the variables. Patients with the combined diagnosis of asthma and COPD were not included into the analysis. An odds ratio smaller than one means that there is a negative association between those variables. Significant results are bold. The logistic regression showed that the diagnosis asthma was often based only on single measurement of PEF (OR 7.6) or 'trial of medication' (OR 8.1). The use of spirometry was not significantly associated with the recorded diagnosis asthma. Concerning the recorded diagnosis of COPD, 'trial of medication' was not associated significantly. Instead, the recorded diagnosis was based on spirometry (OR 4.4). Bronchial challenge testing was used to exclude asthma (OR 6.7). Also chest X-ray showed a significant association with the recorded diagnosis of COPD (OR 4.1). The recording of other lower airway diseases was mainly based on the medical history (OR 10.9), the other diagnostic procedures are used only in few cases (OR < 1). Prescriptions The documentation of the prescribed medication showed that 66% of patients with asthma and 42% with COPD received sympathomimetics (table 3 ). 38% of the asthma patients received inhaled corticosteroids, whereas 46% of the patients with COPD were treated with inhaled corticosteroids. Cromoglycate was given in 36% of patients with asthma and 14% of COPD. Table 3 Relationship between performed diagnostis. actual treatment and documented diagnoses Performed diagnostics and actual treatment Asthma (n = 255) COPD (n = 112) Other (n = 465) n % OR 95% CI n % OR 95% CI n % OR 95% CI Sympathomimetics (n = 167; 65.5%) (n = 47; 42.0%) (n = 36; 7.7%) Medical history only 9 3.5 0.22 0.10–0.51 7 6.3 1.35 0.56–3.26 10 2.2 4.97 2.05–12.02 Medical history and trial of medication 52 20.4 7.99 3.06–20.85 4 3.6 2.93 0.51–16.71 8 1.7 31.48 8.95–111.2 Medical history and spirometry 60 23.5 2.28 1.24–4.18 36 32.1 4.06 1.77–9.35 14 3.0 3.10 1.51–6.36 Medical history and PEF-measurement 46 18.0 0.67 0.43–1.07 0 0 0 0-0 4 1.0 2.39 0.77–7.36 Additional bronchial challenge test 12 4.7 3.51 0.77–16.04 9 8.0 1.16 0.44–3.08 0 0 0.01 0–10 9 Additional chest X-ray 22 8.6 1.02 0.48–2.17 22 19.6 1.98 0.91–4.31 6 1.0 1.91 0.75–4.86 Corticosteroids (n = 96; 37.6%) (n = 52; 46.4%) (n = 28; 6.0%) Medical history only 4 1.6 0.23 0.08–0.66 9 8.0 2.22 0.85–5.78 5 1.0 2.37 0.78–7.23 Medical history and trial of medication 20 7.8 0.91 0.49–1.68 3 2.7 1.16 0.22–6.03 5 1.05 13.99 4.11–47.64 Medical history and spirometry 49 19.2 5.17 2.91–9.19 40 35.7 4.67 2.05–10.64 15 3.2 6.05 2.72–13.45 Medical history and PEF-measurement 23 9.0 0.58 0.33–1.02 0 0 0 0–14 × 10 4 3 1.0 2.26 0.63–8.04 Additional bronchial challenge test 9 3.5 3.30 1.07–10.17 16 14.3 6.22 1.93–20.11 0 0 0.00 0-0 Additional chest X-ray 16 6.3 1.63 0.79–3.37 29 25.9 4.56 2.00–10.38 10 2.2 6.19 2.65–14.47 Cromoglycate (n = 91; 35.7%) (n = 16; 14.3%) (n = 39; 8.4%) Medical history only 9 3.5 0.16 0.07–0.37 1 1.0 0.23 0.05–1.02 21 4.5 12.02 4.93–29.29 Medical history and trial of medication 24 9.4 1.52 0.83–2.79 0 0 0.00 0–1.6 × 10 18 4 1.0 6.16 1.77–21.51 Medical history and spirometry 23 9.0 0.74 0.42–1.32 10 8.9 1.07 0.40–2.86 10 2.2 1.70 0.89–3.25 Medical history and PEF-measurement 35 13.7 1.74 1.01–3.00 5 4.5 14.1 2.96–67.18 4 1.0 2.17 0.71–6.65 Additional bronchial challenge test 5 2.0 1.06 0.34–3.26 4 3.6 1.67 0.48–5.83 1 0 1.62 0.19–13.54 Additional chest X-ray 9 3.5 0.65 0.29–1.46 5 4.5 0.73 0.23–2.25 8 1.7 2.58 1.11–6.01 Only a small amount of patients with 'other lower airway diseases' received anti-obstructive medicine (6–8%). The logistic regression showed that sympathomimetics were given in asthma when having shown efficacy (trial of medication) (OR 8.0) or when spirometry was performed (OR 2.3). Therapy with corticosteroids was significantly associated with spirometry (OR 5.2) and bronchial challenge testing (OR 3.3). Treatment of COPD with sympathomimetics was significantly associated with the use of spirometry (OR 4.1). Inhaled corticosteroids were also given more often when spirometry (OR 4.7) and Chest X-ray (OR 4.6) had been performed. Bronchial challenge testing was used to exclude asthma (OR 6.2). In asthma as well as in COPD corticosteroids are not prescribed only by trial of medication and are not given after making the diagnosis only by medical history. Prescribing of cromoglycate was associated with PEF measurement in both asthma and COPD (OR 1.7 respectively 14.1) Treatment of the other lower airway diseases with sympathomimetics respectively corticosteroids was mainly associated with testing the efficacy of therapy (OR 31.5 respectively 14.0). The prescription is lower associated with spirometry (OR 3.1 respectively 6.1) or x-ray (OR 1.9 respectively 6.2). Cromoglycates were given mostly on the basis of 'taking medical history only' (OR 12.0) and trial of medication (OR 6.2). Discussion Although guidelines recommend the use of spirometry, this was used in only in 30% of the patients with a recorded diagnosis of asthma and in 58% of the patients with a recorded diagnosis of COPD. A high number of recorded diagnoses of asthma and COPD were insufficiently diagnosed by PEF-measurement, medical history and diagnosing by therapy. On the first sight, patients with asthma seem to be treated insufficiently with anti-inflammatory therapy and patients with COPD seemed to be over-treated with steroids. However, further analysis showed a high association between prescribed corticosteroids and sympathomimetics as long term medication and performed diagnostic procedures. This improvement of diagnostic investigation when potential harmful drugs are given demonstrates that treatment of asthma and COPD by the GPs may be more consistent with guidelines than other studies have found [ 10 , 13 ]. This hypothesis is facilitated by the fact, that cromoglycates seem to be administered if there was diagnostic uncertainty, as there was no significant association between performing spirometry and making diagnoses in those cases. According to this, our study shows that a deeper look insight with analysis of diagnoses together with performed diagnostics and medication could prove a better care for people with obstructive airway diseases than a single analysis of the documented diagnoses. As a conclusion, ICD-10 codes seem not to be able to reflect the work in primary care, in particular if the difficulties of the diagnostic and therapeutic differentiation between asthma and COPD in earlier stages are taken into account [ 18 , 20 ]. Therefore it seems that the documented diagnosis due to ICD-10 often serves as a 'working hypothesis'. The needs of family physicians for practice and research would be best met by the International Classification of Primary Care (ICPC-2) [ 24 ], but the general practitioners in Germany are politically obliged to record ICD-10 diagnoses to get reimbursement. As the documentation of the reason of encounter is restricted in these terms of ICD-codes there could be some necessity to assign diagnoses although they do not match with the real status of the patient. This makes the recorded diagnoses unreliable, sometimes resulting in apparently poor coherence between medication and diagnoses, and this leads to difficulties in drawing valid conclusions on the quality of primary care. Another critical point is, that the 'trial of medication' as a 'fact of life in primary care' could implicate a patients' perception of improvement which could lead the doctor to perpetuate the therapy, thus resulting in over-prescribing accompanied by an avoidable risk of side-effects. Especially this 'trial of medication' may reflect diagnostic uncertainty in general practice, which must normally have consequences for the classification and labelling. In absence of a reasonable classification system the GPs are forced to hide this uncertainty by documenting diagnoses in ICD-10-codes. As a result, GPs could feel the uncertainty nevertheless, but start seeing it as a personal shortcoming rather than a reality of medicine. Therefore this exaggeration of 'labelling with ICD-10' could also constrain a systematic quality improvement in diagnostic performance in primary care. The strength of the study was the detailed analysis with manual extraction and documentation of performed diagnostics and actual medication of each identified patient. As the ICD-10 classification made it difficult to identify patients with obstructive airway diseases in junction with performed diagnostics and therapy we searched all records manually. Because of this complexity the study had the limitation that the number of GPs was small, which reduces the generalisability of the descriptive findings, although the estimates of associations are less sensitive for this problem. The prevalence of asthma and COPD in this German sample of general practice seems to be low compared to the prevalence of about 5% in studies of other countries [ 25 - 27 ]. One explanation might be the high doctor-patient contact rate in Germany that decreases the prevalence of severe diseases in general practice. Conclusions The conclusion of this study is that performance assessment should take the reliability problems of recorded diagnoses into account, especially if the ICD-10 classification is used. A more detailed analysis may be necessary for adequate evaluation of primary care, consequential pointing out to the fact, that treatment of obstructive airways diseases by the GP's may be more consistent with guidelines than other studies have found. Despite these findings improvement of diagnostic performance in general practice is necessary, because a routine use of spirometry is of growing importance against the background of the increasing prevalence of obstructive airway diseases. Competing interests The author(s) declare that they have no competing interests. Authors contributions AS conceived and designed the study, wrote the report. LG and IM checked the practice software and documented data. MB helped with interpreting the data and writing the report. MW helped with interpreting the data and writing the report. JS helped with revising the report. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549048.xml |
497052 | Quality of life and emotional distress in advanced prostate cancer survivors undergoing chemotherapy | Prostate cancer continues to occur in over 230,000 men each year. Although the majority of these will be diagnosed in the early stages, there remains a proportion who will either be diagnosed in late stage disease or develop progressive disease. In patients with advanced disease, research has recently focused on using chemotherapy for symptom management and palliation. Given that the focus is not on cure, the effect of chemotherapy on quality of life is of utmost importance. The present article will 1) summarize the current chemotherapeutic studies that have included a quality of life component, with a particular focus on pain and fatigue, 2) discuss the issue of distress in advanced prostate cancer patients treated with chemotherapy, and 3) suggest future research directions. From the studies that have investigated quality of life, it appears that several chemotherapeutic agents reduce pain and fatigue, although the development of fatigue is often the dose-limiting factor of some agents. The assessment of overall quality of life has occurred in several studies, however, an examination into the impact of chemotherapy on functional status and interpersonal relationships has not been studied. Finally, in contrast to the numerous studies in early stage prostate cancer patients, the presence and effect of distress in chemotherapy-treated prostate patients has not been examined. As such, increased attention is needed to quality of life during phase I-III chemotherapy trials. | Background and significance Prostate cancer will be diagnosed in an estimated 230,110 men during 2004 [ 1 ]. There will also be approximately 29,900 men who will die from prostate cancer this year, making it the second leading cause of cancer death among men. While early detection and improved treatments have resulted in improved 5-year survival rates for individuals with early stage prostate cancer (recent data have put the 5-year survival rates at 100% for men diagnosed with local and regional prostate cancer), there remains a proportion of men (roughly 14%) who will be diagnosed with advanced prostate cancer. For these individuals, the 5-year survival rate is much lower. Indeed only 34% of men diagnosed with distant disease will survive for 5 years [ 1 ]. For men with late stage disease, chemotherapy is increasingly being examined as a treatment option, although the goal is usually palliative in nature and may not extend length of survival [ 2 , 3 ]. Chemotherapy is also being explored as adjuvant therapy in men with early stage disease where length of survival may be lengthened by its administration. In both cases, but particularly among men receiving chemotherapy as treatment for advanced cancer, the effect that chemotherapy may have on quality of life (QOL) is extremely important. This QOL includes not only the individual's physical well-being, but their mental well-being, role functioning and levels of emotional distress as well. At present, few studies have examined the impact of chemotherapy on the physical and emotional QOL of prostate cancer patients. The goals of the current article are to: 1) summarize the current chemotherapeutic studies that have included a quality of life component, with a particular focus on pain and fatigue, 2) discuss the issue of distress in advanced prostate cancer patients treated with chemotherapy, and 3) suggest future research directions. Impact of chemotherapy on quality of life The inclusion of QOL endpoints in chemotherapy trials with cancer patients started in earnest during the last decade, with the majority of studies assessing the impact of chemotherapeutic agents on symptoms such as pain and fatigue. Since then, the inclusion of QOL endpoints has become a more common outcome in chemotherapy studies, although many continue to neglect the psychological component, focusing instead on the occurrence of symptoms and their impact on physical QOL. Methods A comprehensive search of English-language articles in PubMed was conducted to identify studies that had assessed quality of life and/or emotional distress as part of chemotherapeutic trials in advanced cancer patients (Table 1 ). The keywords "advanced cancer", "chemotherapy", and "quality of life" were included in each search. In subsequent searches "distress", "anxiety", and "depression" were added. Table 1 Summary of reviewed articles Authors Chemotherapeutic agent QOL measure Direction of effect Tannock et al. (1996) Mitoxantrone EORTC QLQ-30; Pain rating Improved pain, mood & physical activity Beer et al., (2001) Docetaxel Present Pain Intensity scale Improved pain Sinibaldi et al. (1999) Docetaxel Brief Pain Inventory Improved pain Copur et al. (2001) Docetaxel Numeric rating scale Improved pain Gravis et al. (2003) Docetaxel EORTC QLQ-30; Pain VAS Improved role, social functioning, overall QOL, pain, fatigue, constipation Small et al. (2000) Suramin Brief Pain Inventory Improved pain Fuse et al. (1996) Cisplatin/carboplatin Rating scale Improved pain Kreis et al. (1999). Docetaxel Physician-graded events Presence of fatigue dose-limiting factor Hamilton et al. (2003) Vinblastine Physician-graded events Presence of fatigue dose-limiting factor van Andel et al. (2003) Epirubicin QLQ-30 Reduced fatigue, transient improvement in emotional, social and cognitive functioning Kornblith et al. (2001) Docetaxel FACT-P, Mental Health Inventory-17 Transient improvements in emotional well-being, improved overall QOL Pain In one of the first studies to examine the impact of chemotherapy on pain, Tannock, Osoba, Stockler et al. [ 4 ] randomized 161 hormone-resistant prostate cancer patients either to prednisolone or prednisolone plus mitoxantrone. The goal was to determine the impact on pain reduction as a palliative endpoint. Also included was the overall assessment of QOL using the EORTC QLQ-30 and a specific prostate cancer QOL measure composed of nine analog scales. The results demonstrated that the addition of mitoxantrone to prednisolone reduced pain in 29% of patients compared to 10% for those who only received prednisolone. Improvements in pain, mood, and physical activity were also observed on the QOL measures for the individuals who received mitoxantrone. Additional analyses from this study revealed that after six weeks of treatment, pain and physical functioning remained improved in the mitoxantrone plus prednisone group. Moreover, after 12 weeks of treatment, overall quality of life in this group was improved, as was quality of life in four functional domains and nine specific symptoms [ 5 ]. Additional studies with mitoxantrone have demonstrated that up to 40% of patients will report reductions in pain and improvements in QOL [ 6 ]. As a result, in hormone-resistant prostate cancer patients, mitoxantrone is believed to be of some benefit [ 4 ]. Pain was also assessed in several studies utilizing docetaxel either alone or in combination with estramustine in androgen-independent prostate cancer [ 7 - 9 ]. In one study, the administration of docetaxel as a single agent resulted in pain relief, defined as two-point reduction in the Present Pain Intensity scale on two consecutive evaluations, in 30% of patients [ 10 ]. Moreover, an additional 18% reduced their consumption of analgesics by at least 50%. Mean pain scores on quality of life scales were also reduced over the course of treatment [ 7 ]. In two studies using the combination of docetaxel and estramustine, pain reductions were observed in 70% and 82% of patients [ 8 , 9 ]. Unfortunately, the methods used to assess pain are not thoroughly reported in either study. In a recent study, docetaxel was administered intravenously on a daily basis for 6 consecutive weeks followed by a 2-week break between each of up to 4 cycles [ 11 ]. The authors assessed QOL prior to the start of chemotherapy, the first day of each treatment cycle, and then 15 and 30 days after the last treatment. At baseline, metastatic, hormone-refractory prostate cancer patients reported decreased QOL in role, social functioning, and overall QOL, as well as pain, fatigue, and constipation. Of note, following the first cycle, all patients reported improved QOL and pain relative to baseline levels. At the end of treatment, pain levels had increased somewhat, but were still lower than baseline levels. Combined, these studies suggest that docetaxel is effective in reducing pain in androgen-independent patients. Reductions in pain have also been observed in studies using suramin and epirubicin. In a study by Small et al. [ 12 ], comparing suramin plus hydrocortisone to a placebo plus hydrocortisone, reduction in pain for longer durations were observed in the suramin treated group. Interestingly, however, more global measures of QOL were not impacted by that treatment. Despite the reduction in pain, because overall QOL and survival rates are similar to hydrocortisone treated patients, whether suramin should be regularly used in the treatment of hormone-refractory prostate cancer patients is unclear. This is also true for suramin with anthracycline regimens, where the combination, because it does not offer significant improvements in disease management, is not recommended to hormone-resistant prostate cancer patients [ 13 - 18 ]. Pain reduction has also been observed in prostate cancer patients treated with epirubicin [ 19 ]. Interestingly, the reductions in pain did not correspond to patient-reported improvements in QOL. As a result, like suramin, the authors concluded that epirubicin should not be used as a monotherapy. The use of the more commonly known platinum compounds (e.g., cisplatin, carboplatin) have also demonstrated beneficial efforts on pain reduction. In advanced prostate cancer patients treated with a carboplatin/epirubicin/etoposide regimen, pain relief was observed in 44% of the patients [ 20 ]. The overall point of these studies is that for prostate cancer patients with advanced disease, there are a variety of chemotherapeutic agents that have beneficial palliative effects, specifically pain reduction. Despite this, there is no definitive palliative pain regimen in this population and no defined algorithm for pain management using chemotherapy. Fatigue Several studies have investigated the relationship between chemotherapeutic agents and fatigue in prostate cancer patients. Among the agents studied are the taxanes, specifically docetaxel and paclitaxel. In phase I studies of docetaxel and estramustine, the presence of fatigue has been used to identify the dose-limiting factor [ 21 , 22 ]. Currently, a phase III trial is underway to determine the impact on QOL of docetaxel/estramustine compared with mitoxantrone/prednisolone [ 23 ]. In a similar phase I/II study examining the effect of vinblastine for androgen-independent prostate cancer, fatigue was also reported as the dose-limiting factor. More importantly, however, the agent was found to be inactive resulting in the conclusion that vinblastine is not an appropriate treatment [ 24 ]. Fatigue was also an endpoint in a well-designed study that assessed the effect of 25 mg/m2 epirubicin administered intravenously on a weekly basis compared to 100 mg/m2 administered every 4-weeks [ 19 ]. After four weeks of treatment, individuals who were in the Epi100 group reported less fatigue. They also reported better emotional, social, and cognitive functioning as measured by QLQ-30, although these differences disappeared at repeated assessments. The effect of subsequent administration of either dosage of epirubicin on fatigue was not reported, although the authors note that there were no differences at subsequent assessments between groups. As such, it is unknown whether epirubicin decreases or increases fatigue in prostate cancer patients over time. Quality of Life (QOL) In contrast to the numerous QOL studies of early-stage prostate cancer patients, beyond pain and fatigue, the assessment of QOL in advanced stage prostate cancer patients is relatively lacking. By and large there are no studies assessing the common areas of physical well-being (e.g., urinary and sexual functioning) or psychological well-being (e.g., distress related to treatment or sequelae), and social or family well-being in chemotherapy treated patients. The notable exception is a study of the QOL of 44 hormone refractory prostate cancer patients and their partners by Kornblith et al. [ 25 ]. In a phase II study of docetaxel, estramustine, and low dose hydrocortisone, prostate cancer patients were assessed with the Functional Assessment of Cancer Therapy-Prostate, as well as the Mental Health Inventory-17. Over a period of 6-months, men reported significant improvements on the Emotional Well-being subscale. Further examination, however, revealed that when compared to the baseline assessment, emotional well-being was only better at the two and four month assessments. Total FACT-P scores improved over the course of treatment as well, as did prostate cancer-specific concerns, although the former was not significant [ 25 ]. The authors concluded that the benefits of the chemotherapeutic regimen were limited to the first four-months of treatment. Impact of chemotherapy on emotional distress In contrast to the various studies that have investigated the presence of emotional distress, depression, and anxiety in early stage prostate cancer patients, there is remarkably little concerning these issues in those with advanced cancer. Indeed, a Pubmed search using the terms "distress AND advanced prostate cancer" resulted in only 12 studies. Of these, only 3 studies specifically focused on advanced prostate cancer [ 25 - 27 ], with 1 of these focusing on physical symptom distress [ 26 ] and another focused on a group of asymptomatic men with nonmetastatic prostate cancer receiving androgen deprivation therapy [ 27 ]. Only one of the studies, the previously cited Kornblith et al. [ 25 ] study assessed distress in chemotherapy treated men. The result, therefore, is an overwhelming lack of information concerning the emotional functioning of chemotherapy-treated prostate cancer patients. It is hard to believe that these individuals who are frequently androgen-deprived, have a short life expectancy, and are being treated with chemotherapy for palliative purposes are not experiencing some emotional distress. Even as part of phase II studies, where the sample sizes are small, emotional distress should be examined if for no other reason than to provide initial information as to what changes may be seen in a larger sample, and what issues need to be assessed. Conclusions and implications Chemotherapy is being examined with greater frequency in the treatment of advanced prostate cancer. Since the goal of treatment for these individuals is more likely to be palliation than cure, understanding the impact on QOL is that much more important. For example, understanding the impact on QOL can assist clinicians and patients in decision-making by providing data on whether symptom benefit obtained outweighs the toxicities from treatment. Thus far, several studies have identified improvements in pain and fatigue as a result of chemotherapeutic protocols, although in some studies, the presence of fatigue is a dose-limiting factor. Disappointing is the lack of focus on the more global aspects of quality of life, the impact on physical, psychological, social, and family well-being. How the treatment of the individual affects their relationships with others (e.g., marital relationship, sexual functioning) has yet to be studied in this population. Also yet to be studied is the impact on emotional well-being. The presence of distress in cancer patients is well-documented (e.g., [ 28 ]), and it is known that early-stage prostate cancer patients exhibit distress (bother) over physical side effects (i.e., urinary and sexual functioning). In addition, distress related to PSA tests has been documented. What then is the emotional well-being of advanced prostate cancers like? Does the administration of chemotherapy play a role in either the development of distress or its reduction? As the continued development and testing of chemotherapeutic medications for advanced prostate cancer moves from phase I through phase II and III trials, it is of ever-increasing importance to assess the impact on QOL and distress. Only through its inclusion will a clearer picture of the efficacy of chemotherapy be observed. Future research can do this by developing protocols that include HRQOL parameters as a standard part of the design process. Both the industry that develops and tests the compound and the academics who deliver it to the patient should be involved in identifying which aspects of QOL are essential to target. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC497052.xml |
521486 | PEDRo: A database for storing, searching and disseminating experimental proteomics data | Background Proteomics is rapidly evolving into a high-throughput technology, in which substantial and systematic studies are conducted on samples from a wide range of physiological, developmental, or pathological conditions. Reference maps from 2D gels are widely circulated. However, there is, as yet, no formally accepted standard representation to support the sharing of proteomics data, and little systematic dissemination of comprehensive proteomic data sets. Results This paper describes the design, implementation and use of a P roteome E xperimental D ata R ep o sitory (PEDRo), which makes comprehensive proteomics data sets available for browsing, searching and downloading. It is also serves to extend the debate on the level of detail at which proteomics data should be captured, the sorts of facilities that should be provided by proteome data management systems, and the techniques by which such facilities can be made available. Conclusions The PEDRo database provides access to a collection of comprehensive descriptions of experimental data sets in proteomics. Not only are these data sets interesting in and of themselves, they also provide a useful early validation of the PEDRo data model, which has served as a starting point for the ongoing standardisation activity through the Proteome Standards Initiative of the Human Proteome Organisation. | Background Bioinformatics tools and techniques depend directly or indirectly upon experimental data. However, interpreting experimental data often requires access to significant amounts of additional information about the sample used in the experiment, the conditions in which measurements were taken, the equipment used to take the measurements, etc. Recent proposals for models that capture such experimental descriptions alongside experimental results include MIAME for transcriptome data [ 1 ] and PEDRo for proteome data [ 2 ]. However, if full use is to be made of such rich data models for genomic data, these models must also be associated with comprehensive software tools for data capture, dissemination and analysis. In proteomics, which is rapidly evolving into a high-throughput experimental approach, there is (as yet) no standard representation for experimental data. As a result, limited tool support is available for disseminating, searching, comparing or analysing the results of experiments conducted using different techniques and equipment in different laboratories. Thus, while experimental results can be analysed, often in a labour-intensive manner in-house, the development of bioinformatics techniques for archiving, sharing and wider exploitation of proteomics results is still in its infancy. This paper seeks to contribute to the development of effective and systematic support for proteome data management by: 1. Describing a database for storing, searching and disseminating experimental proteomics data. This material should be relevant to the developers of future proteome data management systems in that it discusses and illustrates various design and implementation decisions that have an impact on the role and maintenance of the resulting database. 2. Making available data sets from several labs whose data have been included in the initial release of the database. These data sets themselves result from substantial experimental activities, and are representative of the sorts of information that in-house and public proteome data repositories must capture. As the database stores data in an XML format that conforms to the PEDRo ( P roteomics E xperimental D ata R ep o sitory) data model [ 2 ], this material provides concrete examples for other users of data that conform to this schema, and should be useful for validation of specific parts of the model as input to the Human Proteome Organisation Proteome Standards Initiative (HUPO-PSI) activity on models for proteome data [ 3 ]. The database described in this paper has similar objectives and functionality to various other databases for functional genomic data. In particular, like the Gene Expression Omnibus (GEO) [ 4 ], the Stanford Microarray Database [ 5 ] and ArrayExpress [ 6 ], it contains a single category of experimental data, while accommodating the production of that data using several different experimental techniques. Like ArrayExpress, and unlike GEO, for example, the data stored in PEDRo must conform to a rich, but nevertheless deliberately constraining, data model. This model is richer than that supported by the well established SWISS-2DPAGE database [ 7 ] in that it not only contains information on protein separation and identification, but also includes detailed descriptions of experimental samples, the mass spectrometric analyses conducted, and the software used to perform protein identifications. Establishing the most appropriate kinds of data to include in a database such as PEDRo is not straightforward, as this depends on the use that is to be made of the data. In a large data repository, users may want to search for results based on widely varying criteria – for example, the proteins identified, the change in the level of a protein over time, the mechanism by which a sample was studied, etc. Furthermore, the users of a proteome data repository may themselves be diverse, and include: experimentalists with minimal direct experience of proteomics, but who are interested in proteins or organisms for which proteome studies have been conducted; proteome scientists who want to identify how successful specific techniques have been in different contexts; or mass-spectrometric analysts who want to compare their results with those of others. This wide range of potential users encourages the creation of a rich repository for proteomics data that provides detailed descriptions of many different aspects of an experiment. However, populating a database such as PEDRo is not a trivial task, as several of the different kinds of data included in PEDRo currently have to be entered manually, which is time-consuming for data providers. Even though a data entry tool has been developed to ease data entry (available from [ 8 ]), experience populating the database suggests that the creation of a data set from scratch (e.g., for a sample analysed using a single gel, for which multiple identifications have taken place) can take around a week, but that creating subsequent data sets that share some aspects of the experimental set-up is significantly less time-consuming. In addition, widespread deployment of a standard model should lead to laboratory equipment, or associated software, producing data that conforms to the standard, so the longer-term position for high-throughput laboratories should involve much lower data capture costs. It is hoped that the early provision of a collection of data sets conforming to the widely discussed PEDRo model will be useful in informing ongoing activities on the HUPO-PSI proteome data standard [ 3 ]. Construction and content Many bioinformatics databases, such as UniProt [ 9 ] and PDB [ 10 ], are associated with file formats that can be parsed by software that analyses or displays the data from the database. The Extensible Markup Language (XML [ 11 ]) has been developed in part to make the description, parsing and display of such files more systematic; thus there is a trend in bioinformatics towards the use of XML for storing or transmitting biological data [ 1 , 2 ]. The PEDRo database makes extensive use of XML for capturing, transmitting, storing and searching proteomics data. In particular: 1. The data-capture process uses a software tool, illustrated in Figure 1 , which prompts users for values for different fields, and includes facilities for importing substantial data files, such as those representing peak lists. The tool constructs data-entry forms from the XML Schema definition of the PEDRo model. An XML Schema describes the structure of an XML document, and thus makes explicit the hierarchical structure of the document, the elements that are contained within the document, the types of those elements, and the number of times different elements may occur. The result of the data capture process is thus an XML file that corresponds to the PEDRo schema. A fragment of the XML format for a PEDRo entry is provided in Figure 2 . Figure 1 The Pedro data capture tool. The Pedro data capture tool in use, editing a proteome data file. The left hand panel provides a tree-based browser for the complete document, while the right hand panel supports data entry for a specific component of the model, in this case a sample . The data capture tool is available for download from [8]. Figure 2 Sample PEDRo XML. A fragment of XML for an S. cerevisiae sample. The XML Schema from the model, plus complete data sets for the experiments described in Table 1, are available from [8]. 2. The database stores the XML captured using the data entry tool directly, using Xindice [ 12 ], an open-source XML storage system. Several different storage options exist for XML data, including: (i) storing the XML directly in a native XML repository such as Xindice; (ii) storing the XML directly using the XML storage extensions provided by commercial relational database vendors; and (iii) mapping from the XML documents onto tables for storage in a relational database. We have chosen option (i) for PEDRo. Option (ii) was not adopted because there is not yet a standard for integrating XML storage with relational databases, although this is being developed [ 13 ]. Option (iii) was not adopted because we envisage that the data model used in PEDRo will evolve to reflect the HUPO-PSI standard [ 3 ], and we wanted to avoid the need to evolve both relational and XML versions of the database in parallel. Furthermore, the emphasis for the PEDRo database is on enabling users to identify relevant experimental data sets, rather than on conducting complex searches or analyses over such data sets. For the required tasks, the query facilities provided with XML databases such as Xindice, which tend for the meantime to be based upon XPath [ 14 ] are sufficient. 3. The data are presented to users by generating web pages from the stored XML using XSLT [ 15 ], which was designed to support exactly this sort of task. This means that it has been straightforward to develop reports from the stored form of the data. Furthermore, the download format for the data is as XML documents, in the hope that this will ease the development of tools for parsing and analysing data obtained from the database. The software components used within PEDRo (and the role they play in data capture, storage and dissemination) are illustrated in Figure 3 . Figure 3 PEDRo software components. The software components used in PEDRo. In essence, data flows clockwise from the top left, with three categories of user. The first category of user is the scientist who carries out data entry – this user must be intimately familiar with the experiment that has been conducted and the equipment that has been used. The result of the data capture process is a PEDRo XML file. This XML file is then checked by the database curator, who can then add the data into the database. Once in the database, the PEDRo Database Access software can be used to search the database and view its contents. The PEDRo software has been implemented over the Xindice XML database [12] using Java Server Pages [28]. The data stored in the database for each experiment is as described in the PEDRo model [ 2 ], and thus involves sample generation (e.g. organism, growth conditions, tagging), sample processing (e.g. gel properties, spot details), mass spectrometry (e.g. machine settings, peak lists) and in silico analyses (e.g. database search program used and results obtained). Table 1 provides a high-level overview of the initial data sets in the database. The data in the initial release of the database illustrates several different proteomics techniques in use, including sample processing based on classical 2D Gels and DIGE, the use of different gel imaging software, mass spectrometry using MALDI-TOF and MS/MS, and in silico data analyses using more than one program. Furthermore, the data captured covers a range of different organisms, including Saccharomyces cerevisiae , Candida albicans , Candida glabrata , Mus musculus , Arabidopsis thaliana and Streptomyces coelicolor . Table 1 Summary of database contents. A summary of the data sets included in the initial release of PEDRo. The database provides more detailed descriptions of sample generation , sample processing , mass spectrometry and in silico analyses , populating the model described in [2]. 1 Organism Saccharomyces cerevisiae Sample Generation Whole cell extracts (bead beating) Experimenter Al Brown et al. Sample Processing 2D gel Description GCN4-dependent proteins that respond to amino acid starvation Mass Spectrometry MALDI-ToF In Silico Analysis Peptide mass fingerprint searches (MASCOT, MS-Fit) 2 Organism Candida albicans Sample Generation Whole cell extracts (bead beating) Experimenter Al Brown et al. Sample Processing 2D gel Description GCN4-dependent proteins that respond to amino acid starvation Mass Spectrometry MALDI-ToF In Silico Analysis Peptide mass fingerprint searches (MASCOT, MS-Fit) 3 Organism Candida glabrata Sample Generation Whole cell extracts (bead beating) Experimenter Ken Haynes et al. Sample Processing 2D gel Description Proteins that respond to the inactivation of ACE2 Mass Spectrometry MALDI-ToF In Silico Analysis Peptide mass fingerprint searches (MASCOT, MS-Fit) 4 Organism Streptomyces coelicolor Sample Generation Whole cell extracts (sonication) Experimenter Andrew Hesketh Sample Processing 2D gel Description Changes in the proteome of strain M600 during growth and antibiotic production in liquid medium Mass Spectrometry MALDI-ToF In Silico Analysis Peptide mass fingerprint searches (MASCOT) 5 Organism Mus Musculus BALB/C Sample Generation Exfoliated jejunal epithelium prepared as per Bjerknes & Cheng, Anat Rec 1981, 199, 565. Experimenter Alan Pemberton, Pamela Knight Sample Processing 2D gel Description Trichinella spiralis infection in mice induces alterations in the proteome of the small mucosal epithelium Mass Spectrometry MALDI-ToF In Silico Analysis Peptide mass fingerprint searches (MASCOT, MS-FIT) 6 Organism Homo sapiens Sample Generation Gel image analysis data from 12 patients, used to assign spot picks from a preparative 2D gel Experimenter Tony Whetton, Caroline Evans Sample Processing 2D gel Description Proteomics can identify prognostic markers in CLL disease progression Mass Spectrometry MALDI-ToF In Silico Analysis Peptide mass fingerprint searches (MASCOT) 7 Organism Saccharomyces cerevisiae Sample Generation Saccharomyces cerevisiae , strain YMK36, butanol – and butanol + Experimenter Kathleen Carroll Sample Processing 2D gel Description Effect of butanol stress on yeast Mass Spectrometry MALDI-ToF In Silico Analysis Peptide mass fingerprint searches (MASCOT) 8 Organism Saccharomyces cerevisiae Sample Generation Experimenter Kathleen Carroll Sample Processing 2D gel Description Mapping Heat shock proteins in Saccharomyces cerevisiae Mass Spectrometry MALDI-ToF In Silico Analysis Peptide mass fingerprint searches (MASCOT) 9 Organism T. bruceii Sample Generation Gel pieces Experimenter Sarah Hart Sample Processing Bands from 1D gel Description Trypanosome Flagella Mass Spectrometry MALDI-ToF, LC MS/MS In Silico Analysis Mascot MS/MS These PEDRo data are significant in biological terms. For example, they include the first direct comparison of proteomic responses in two fungal species, namely responses to amino-acid starvation in the baker's yeast S. cerevisiae and the pathogenic fungus, C. albicans [ 16 ]. In addition, they include the first proteomic analysis of the medically important pathogen C. glabrata (Stead et al ., Proteomic changes associated with inactivation of the Candida glabrata ACE2 virulence-moderating gene, manuscript submitted), whose genome sequence has only just been completed. The Streptomyces coelicolor M600 data set is the largest proteomics time course analysis of this strain in terms of numbers of proteins identified. It adds significantly to our knowledge of expression of some of the 20 gene sets annotated as being determinants of the biosynthesis of secondary metabolites, including antibiotics. Somewhat similar experiments, but differing in many aspects of their metadata, are reported on the SWICZ database [ 17 ]. This provides an opportunity to evaluate PEDRo in the context of related data presented in different databases. Also included are data from an experiment investigating the proteomic analysis of the mouse jejunal epithelium and its response to infection with the intestinal nematode, Trichinella spiralis [ 18 ]. Utility Web-based interfaces to biological databases tend to support one or more of the following tasks: browsing – interactively listing or navigating through database entries; searching – identifying database entries on the basis of simple restrictions on the values of one or more fields; visualising – presenting a visual representation of the data as a starting point for browsing; or querying – specifying a search that is to be conducted over the database using a query building interface or by providing inputs to pre-written (or "canned") queries. Functional genomics databases tend to emphasise browsing and searching. For example, the Stanford Microarray Database [ 5 ] supports browsing based around organisms and experiments, and more complex Boolean searches based on criteria such as experimenter, organism and category of experiment. ArrayExpress [ 6 ] supports browsing through experiments, arrays and protocols, and searching based on criteria such as species, experiment type and author. SWISS-2DPAGE [ 7 ] supports browsing by clicking on spots on gels, and searching based on criteria such as description, accession number or author. PEDRo also emphasises browsing and searching. Figures 4 and 5 illustrate the web-based interface to PEDRo, which can be accessed at [ 8 ]. In essence, the records in the database can be accessed by browsing summaries of the entries in the database, or by searching using one or more criteria. These criteria were obtained through a systematic requirements analysis with potential users from several different research groups, who were asked to comment on early versions of the interface. Overall, PEDRo provides core data access facilities that are principally intended to allow users to identify data sets that are of interest to them. As such, the PEDRo database as described should not be seen as a comprehensive query or analysis environment for proteomics data, but rather as a repository through which experimental results can be made available to a wider community. Therefore, S. cerevisiae data from PEDRo will also be made available through GIMS [ 19 ], for example, to enable the integration of these data with other sequence and functional information. Figure 4 Search page. Searching the PEDRo database. The search facility is intended to support rapid identification of PEDRo entries of interest. Searching can be conducted on one or more of the species name, experimenter, hypothesis, gene name and ORF number. Where more than one value is provided, entries are retrieved that match all the values given. In all cases, matches can be partial. Thus, for example, typing "Sacc" into the Species Name field will retrieve entries for Saccharomyces cerevisiae . Figure 5 Results page. Viewing results of the PEDRo search from Figure 4. Discussion A significant motivating factor behind the development of the PEDRo repository has been to allow informed discussion, assisted by concrete examples, into the level of detail and forms of model that are most appropriate for a proteome data repository. As the PEDRo model is being used as the starting point for the HUPO-PSI activity on models for proteome data, early validation of this model is important. The following observations have been made about the PEDRo model during the data capture process: 1. Sample description is neither very precise nor systematic . The effective description of samples is an open issue that spans different kinds of functional genomic data. For example, work is underway on the development of an ontology for characterising microarray experiments, focusing, in particular, on samples [ 20 ]. However, as the variety of organisms, genetic manipulations, extraction techniques, environmental conditions and experimental manipulations that may characterise a sample are extremely large, a mature solution to this problem may be some way off. 2. There is only limited support for relative protein abundance data (e.g. DIGE and stable isotope labelling strategies) . Thus, for example, there is no place in the model to describe an expression ratio for a protein species derived from quantitative experimental strategies, only the ability to capture the 'raw' numbers. In fact, the PEDRo model was not designed to capture expression ratios, partly because such numbers are easily derived from the captured primary data, and partly because the particular method of their derivation may be contentious. It is hoped that the HUPO-PSI model will provide generic constructs for representing relationships between certain kinds of measurement (e.g. relative protein expression readings), to which can be attached the specific detail for individual techniques. However, it also seems important to avoid the pitfalls associated with overly permissive models, as these provide a less stable foundation for the developers of analytical tools than their more proscriptive counterparts. 3. The gel model is not particularly detailed . Thus, for example, there is no detailed description of the image analysis software used, the descriptions of individual spots are fairly minimal, and no details are captured on spot excision. An earlier critique of the PEDRo model for gels, and some possible extensions, is provided by [ 21 ]. It seems that, in order to provide insights for the developers of gel-based experiments, it would be appropriate for the model to be revised to provide additional details on gels. Overall, the appropriate level of detail for a proteomics repository is somewhat subjective, but can usefully be based on guiding principles; agreement as to the principles should then avoid scope-based discussions at a very fine-grained level. The current PEDRo model essentially supports the principle that enough detail should be captured about an experiment to: i. Allow results of different experiments to be analysed/compared. ii. Allow suitability of experiment design and implementation decisions to be assessed. iii. Allow protein identifications to be re-run in the future with new databases or software. There is also an additional negative principle, to the effect that the model itself should not be designed to include dependencies on characteristics relating to the configuration or properties of an individual piece of equipment. Accordingly, we have attempted to allow experimental methods and results to be described in significant detail, but without including parameters and properties that are likely to be superseded rapidly when new models of equipment are introduced, and without including parameters that can only be understood with reference to the documentation of a particular product. The data stored in PEDRo is more comprehensive for each experiment than is the case for most existing proteome databases. For example, in the longest established experimental proteomics resource, SWISS-2DPAGE [ 7 ], the emphasis is on annotated gels, and there is much less information collected on how the annotations were arrived at. Furthermore, there is an architectural distinction – SWISS-2DPAGE follows a more federated approach, with individual sites continuing to hold their own data. These other proteome data sources can be accessed through WORLD-2DPAGE, a web resource listing sites making available experimental proteomics data [ 22 ]. An example of a database that participates in WORLD-2DPAGE is the University of Alabama (UAB) Proteomics Database [ 23 ] which provides search and browsing facilities over data from its host university. As such, the emphasis is on annotated gels, and relatively few details are captured on sample processing, mass spectrometry or in silico analysis. Such design decisions are appropriate for certain categories of user of a proteomics database, but not for others. The UAB database has been designed to provide access to processed experimental results for biomedical researchers, but does not provide enough information to allow detailed comparisons of the ways in which the results were obtained. The ProteomeWeb [ 24 ] provides a wider range of tools than PEDRo (for example, for computing theoretical maps), and supports browsing of annotated gels from several bacteria and archaea. Once again, though, the data provided for each experiment are less comprehensive than in PEDRo. ProDB [ 25 ] has a certain amount in common with UAB, in that it too provides search and browsing over a database of locally produced data. In addition, ProDB features an architecture that supports the plugging-in of data-loading and analysis tools. However, the level of detail supported by the model is not obvious from the paper, which gives only part of the model, and the database was not publicly accessible at the time of writing. In consisting of a collection of tools associated with a database, ProDB thus also has a certain amount in common with SBEAMS [ 26 ] which includes a relational database of proteomic data. The SBEAMS model emphasises the description and analysis of mass spectrometry data, but seems not to support open access to experimental data at the time of writing. In terms of quantities of data, there are fewer data sets in PEDRo than in SWISS-2DPAGE, reflecting the fact that PEDRo is a newly created resource (Release 16 of SWISS-2DPAGE contains 34 reference maps), but somewhat more than in the UAB Proteomics Database. The Open Proteomics Database (OPD) supports the browsing and downloading of comparable amounts of data to those in PEDRo, and also includes mass spectrometry data, although quite a lot of the data are in flat-file format [ 27 ]. However, it is fair to say that none of the current databases is operating in the context of high-throughput experimentation, which will certainly be prevalent in the near future. Conclusions The need for wider and more systematic dissemination of experimental proteomics data is widely recognised, as argued in [ 27 ], and attested to by the ongoing work of the Proteome Standards Initiative [ 3 ]. As such, issues that need to be addressed include: i. The nature and variety of information that should be recorded about proteomics experiments. ii. The functionality that should be provided by repositories that make large-scale proteomic data available. iii. The computational architecture that should be used to provide the functionality at (ii). iv. The nature of the tools that should be developed for use with such a repository. This paper has sought to address issues (i), (ii) and (iii), with a particular emphasis on (i). Following on from [ 2 ] we believe that the provision of a collection of representative proteomic data sets conforming to a consistent model is important to the ongoing process of developing a stable and effective de jure standard for proteome data representation and sharing. This paper describes a database that includes a rich collection of representative data sets. Furthermore, the paper describes the functionality (issue ii) and architecture (issue iii) of an exploratory system for disseminating such data. In the same way as we see models for representing proteomic data evolving in the light of practical experience, we anticipate that the PEDRo repository, and the overall understanding of the data access and dissemination requirements for proteomic data, will evolve as the opportunities presented by high-throughput experimental techniques and comprehensive data sets become more fully understood. Availability and requirements The database can be accessed using a web browser at , by following the Database link. Authors' contributions KG and CG implemented the software. SJ, NM and CFT contributed to the development of earlier prototypes. TM coordinated the data capture activity. CK, CE, AW, SH, DS, ZY, AJPB, AH, KC, LH, MM, PG, JH, KSL, SJG conducted or led experimental activities that generated the data in the database, and contributed to feedback on the model. AB, SJH, SJO and NWP oversaw the database design and development activity, and the latter led the write-up. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521486.xml |
543571 | Review of "Fundamentals of Clinical Trials" by LM. Friedman, CD. Furberg and DL. DeMets | null | Clinical Research faces perhaps a certain grain of confusion. Often, it is mistakenly reduced to case report. Caveats of even greater risks may be hidden in the secrets of patents and interests of manufacturers covertly promoting their products. Thus, a book clearly stating what a clinical trial is, well located within the scientific frame, appears as a significant contribution enhancing the importance and complexity of XXIst Century human health. The book is preceded by two editions (1981 and 1985) and well backed by the experience and prestige of its authors. Clinical Research , a branch of Applied Research , finds in clinical trials the most definitive tool for evaluation of its actual applicability. The book is divided in 19 chapters, each setting up unequivocally the Fundamental Point to be treated, as for example the introductory Chapter 1: A properly planned and executed clinical trial is a powerful experimental technique for assessing the effectiveness of an intervention . A clinical trial compares the effect and value of intervention(s) against a control in human beings. Intervention refers either to a drug, a procedure or a technological device, thus implying that biomedical engineering scientists and professionals might be involved in it. Clinical Research always presents early stages where the laboratory work is mandatory. This book emphasizes the concept all along, so placing the clinical trial in its proper hierarchical perspective. There is laboratory work during drug, device and/or procedure development, animal studies and early tests in small number of human beings. Case reports, typical of the hospital, find their place as a warning of adverse effects or as a flag of an unexpected beneficial effect. In Chapter 2 the authors emphasize that a clinical trial must have a primary question. It should be carefully selected, as any subsidiary question must be, too. Otherwise, the investigators may lose the track. Chapter 3 deals with the study population, underlying the importance of unambiguous eligibility criteria, the latter having a direct influence on the recruitment of participants and, thus, on the final number involved in the project (which may end up with not enough data). Chapter 4 outlines the basic study design; it stresses the demand of a control group and the need of randomization for assigning participants to control and intervention groups. An extremely important subject is the randomization process, essential to avoid any possible bias (Chapter 5). Several forms are presented: simple, blocked and stratified. More elaborate procedures make use of adaptive methods. There are examples and an appendix with an algorithm of general applicability. Bias (a systematic error) is one of the main concerns; thus, a double-blind design is essential (Chapter 6). Chapter 7, Sample Size , addresses the question of how many subjects is enough. The trial must have sufficient statistical power to detect differences between groups. Therefore, calculation of sample size with provision for adequate levels of significance is essential. Several situations with their respective examples are presented making of this pages an invaluable tool. Unfortunately, several equations have been printed in such a way that there is confusion on whether some parameters are factors or exponents. Baseline refers to the status of participants before the start of the intervention (Chapter 8), while Chapter 9 deals with the recruitment of participants. The latter depends on developing a careful plan with multiple strategies. Chapter 10 enters into data collection and quality control, wisely underlining that no study can be better than the quality of its data. Monitoring and even auditing are thus necessary. The history of new medical interventions abounds in sad experiences. Hence, adverse effects, if any at all, must be searched and assessed. Chapter 11 clearly probes into this complex aspect. Sadly, perhaps due to sheer enthusiasm (and I do no want to hint possible commercial interests because the idea alone crosses a risky line), often it has played a secondary role in clinical trials, as stated by the authors. Chapter 12, by Michelle J. Naughton and Sally A. Shumaker, catches the assessment of health-related quality of life, that is, trying to elucidate if the intervention improved, say, physical, psychological and social functioning, perception of well-being and health status, personal productivity, sexual life, sleep disturbances and other parameters. Not an easy task, indeed, although in certain extreme situations (as when life is supported by mechanical means and conscience is no longer present) may lead into deep controversies. The remaining seven chapters deal, respectively, with participant adherence to the program, survival analysis, monitoring response variables (37 pages), issues in data analysis (38 pages full of statistical considerations), closeout (that is, how to properly terminate the study), reporting and interpreting of results (one of the most difficult steps because recommendations will be produced that may end up in massive use of the intervention), and finally a chapter devoted to multicenter trials. The book provides an exceedingly good collection of references so that the interested reader can deepen in a particular subject. All in all, I would highly recommend it, to the physician, basic investigator and biomedical engineer. For the beginner, instead, it may be a little hard for some parts require good knowledge of statistics. It is no doubt excellent material for a course. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC543571.xml |
551607 | Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay | Background In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay. Methods A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes ( set1A , set1B , ial and ipaH ) in Shigella spp. One hundred and ten Malaysian strains (1997–2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth. Results The ipaH sequence was present in all the strains, while each of the set1A , set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non- Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation. Conclusions The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen. | Background Members of the genus Shigella , namely S. flexneri , S. dysenteriae , S. sonnei and S. boydii have caused and continue to be responsible for mortality and/or morbidity in high risk populations such as children under five years of age, senior citizens, toddlers in day-care centres, patients in custodial institutions, homosexual men and, war- and famine-engulfed people. Yearly episodes of shigellosis globally have been estimated to be 164.7 million and of these, 163.2 million were in developing countries and the remaining in industrialized nations. The mortality rate was approximately 0.7% [ 1 ]. A recent study by Lee & Puthucheary [ 2 ] on bacterial enteropathogens in childhood diarrhoea in a Malaysian urban hospital showed that Shigella spp. was the third most common bacteria isolated. S. flexneri and S. dysenteriae type 1 infections are usually characterized by frequent passage of small amounts of stool and mucus or blood. At times, watery stool followed by typical dysenteric stool maybe present with S. dysenteriae type 1 infection. S. sonnei and S. boydii infections are less severe with watery faeces but little mucus or blood. Shigellosis is usually a self-limiting infection, however when it subsides, the intestinal ulcers heal with scar tissue formation. Uncomplicated recovery is usual and the organisms rarely cause other types of infections. Adversely, in 3 to 50% of cases, depending on the virulence of the strain, the nutritional and immune status of the host, the initial infection maybe followed by neurological complications or kidney failure. Serious complications do occur at greatest frequencies in malnourished infants, toddlers, older adults and immunocompromised individuals [ 3 , 4 ]. Virulence genes responsible for the pathogenesis of shigellosis may be located in the chromosome or on the inv plasmid borne by the organism. They are often multifactorial and coordinately regulated, and the genes tend to be clustered in the genome. Previously reported PCR-based detection methods concentrated mainly on the ipaH gene alone [ 5 , 6 ] or on ipaH and ial genes in two separate PCR assays [ 7 , 8 ]. As ial is found on the large inv plasmid which is prone to loss or deletions, this gene-based detection may give false negative results. ipaH , on the other hand, is present on both the Shigella chromosome and on a large plasmid and hence, it is a more stable gene to detect. However, the sole presence of ipaH is not an absolute indicator of virulence as loss or deletion of the plasmid renders the bacterium noninvasive and therefore, avirulent. set1A and set1B are chromosomal genes encoding Shigella enterotoxin 1 (ShET1), which cause the watery phase of diarrhoea in shigellosis [ 9 , 10 ]. ial and ipaH are responsible for directing epithelial cell penetration by the bacterium and for the modification of host response to infection, respectively [ 11 - 13 ]. Here, we describe the application of a multiplex PCR (mPCR) design for simultaneous detection of four virulence genes ( set1A , set1B , ial and ipaH ) in Shigella spp. and to determine the prevalence of these virulence genes in a random selection of Malaysian Shigella strains. Methods Bacterial strains and growth conditions A total of 110 Shigella strains of S. flexneri (n = 84), S. sonnei (n = 15), S. dysenteriae (n = 10) and S. boydii (n = 1) were used in this study. These strains were isolated from patients with diarrhoea in Peninsular Malaysia from 1997–2000, and were provided by the Institute for Medical Research (IMR), Malaysia. Serotyping of the strains ( Shigella antisera from Mast Diagnostics, UK) was carried out by the Bacteriological Unit, IMR. All the strains were checked on Salmonella-Shigella (SS) agar before being transferred to Luria Bertani (LB) agar plate, incubated overnight at 37°C for subsequent screening of virulence-associated genes. All strains were stored at -20°C in LB broth containing 15% glycerol. Development of mPCR Boiled suspension of bacterial cells was used as DNA template. Previously described primers, obtained from Integrated DNA Techs, USA, for detection of the four virulence genes were applied to the template [ 8 , 14 , 15 ] (Table 1 ). Prior to combining all the four primer sets in an mPCR, each pair of primers was optimized singly in separate PCR assays. A typical 25-μl PCR reaction mixture for every primer set consisted of 1x PCR buffer B (Promega, USA), 4 mM MgCl 2 , 130 μM of each deoxynucleotide (dNTP), 0.5 μM of each primer, 1 U of Taq DNA polymerase (Promega, USA) and 2 μl of DNA template. Amplifications were carried out using a Robocycler Gradient 40 Temperature Cycler (Strategene Cloning Systems, USA). The cycling conditions were an initial denaturation at 95°C for 5 min, template denaturation at 95°C for 50 s, annealing at 55°C for 1.5 min, and extension at 72°C for 2 min for a total of 30 cycles, with a final extension at 72°C for 7 min. Table 1 Primers used to identify various virulence-associated genes of Shigella spp. Primer Virulence gene Nucleotide sequences (5' → 3') Size of amplicon (bp) Reference ShET1A set1A TCA CGC TAC CAT CAA AGA TAT CCC CCT TTG GTG GTA 309 14 ShET1B set1B GTG AAC CTG CTG CCG ATA TC ATT TGT GGA TAA AAA TGA CG 147 14 ial ial CTG GAT GGT ATG GTG AGG GGA GGC CAA CAA TTA TTT CC 320 15 Shig1 ipaH TGG AAA AAC TCA GTG CCT CT 423 8 Shig2 CCA GTC CGT AAA TTC ATT CT Based on the results of individual priming, an mPCR was designed. Various parameters such as concentrations of primers (0.5–0.8 μM), MgCl 2 (2 to 4 μM), Taq DNA polymerase (0.6 to 4 U) and dNTPs (100–150 μM) and buffer strength (1.4X to 2.4X) were tested. The simultaneous gene amplifications were performed in a reaction volume of 25 μl consisting of 1.8X PCR buffer B (Promega, USA), 4 mM MgCl 2 , 130 μM of each dNTP, 0.3 μM of each ShET1B primer, Shig1 and Shig2 primers, 0.5 μM of each ShET1A and ial primers, 1 U of Taq DNA polymerase (Promega, USA) and 2 μl of DNA template. All the reaction mixtures were overlaid with 20 μl of sterile mineral oil. Amplifications were similarly carried out as above. After initial screening, strain TH13/00 ( S. flexneri 2a) was chosen as a positive control for PCR assays. A negative control using sterile distilled water as template was included in every PCR assay. The DNA fragments were separated in 2% agarose gel. Reproducibility test The mPCR assay was repeated at least twice with 28 strains to determine the reproducibility of the results, whereby the DNA template of a particular strain was freshly prepared for each repeat. Specificity test The specificity of the mPCR assay was tested with 12 other non- Shigella pathogens: Enterobacter cloacae , Salmonella Paratyphi A (ATCC 9281), S . Paratyphi C, S. Typhimurium, S. Enteritidis, S . Typhi (ATCC 7251), Listeria monocytogenes , Pseudomonas aeruginosa , Klebsiella pneumoniae , Citrobacter freundii , Escherichia coli O157:H7 and E. coli O78:H11. Faecal spiking and sensitivity test This was based on a modification of that described by Chiu and Ou [ 16 ]. Approximately 0.2 g of faeces from a healthy individual was suspended in 1 ml of brain heart infusion (BHI) (Oxoid Ltd., UK) and diluted 10-fold. Then, 1 ml of the diluted faecal suspension was inoculated into 4 ml of BHI and vortexed to obtain a homogenous mixture of broth-faecal suspension. Meanwhile, an overnight culture of S. flexneri 2a was harvested and serially diluted 10-fold with BHI. Then, 250 μl of each dilution of cell culture was mixed with 250 μl of the broth-faecal suspension and 500 μl of BHI in a new eppendorf tube. The tubes were vortexed and preincubated at 37°C for 4 h without shaking. Simultaneously, 100 μl of each diluted culture was plated on LB agar (Oxoid Ltd., UK) to determine the number of viable bacteria in each dilution. After preincubation, mPCR assay was performed on the boiled lysates of each diluted culture. A pure culture of S. flexneri 2a (TH13/00) and an unspiked faecal sample served as positive and negative controls.. The test was repeated with a spiked faecal sample of another healthy individual, and the average detection limit was reported. Screening of clinical specimens 0.2 g of each faecal sample from 10 patients suffering from diarrhoea in a local tertiary University Hospital was suspended in 1 ml of BHI and diluted 10-fold. A volume of 250 μl of broth-faecal suspension was inoculated into 5 ml of BHI and preincubated at 37°C for 4 h without shaking. Concurrently, 100 μl of the suspension was plated onto MacConkey and SS agar plates and incubated overnight at 37°C. mPCR assay was performed on the boiled lysate of the broth-faecal suspension after preincubation. A pure culture of strain TH13/00 and a Shigella -spiked faecal sample served as a positive control, whilst a PCR reaction mixture without bacterial DNA template and an unspiked faecal sample from a healthy individual acted as a negative control. Results Optimization strategies A monoplex PCR for each primer set was initially carried out based on a published report [ 17 ]. Although the concentrations of MgCl 2 (3 mM), dNTP (400 μM each) and primers (1 μM each) were used as recommended, unspecific bands were present together with intense primer-dimers. In order to reduce the background noise and primer-dimers, concentrations of 0.5 μM of each primer and 200 μM of each dNTP were used. Further optimizations of MgCl 2 concentrations (2 to 4 μM) and dNTP (100,130 and 150 μM each) gave intense amplicons with a clean background in each monoplex amplification (Fig 1 , lanes 1–4). Figure 1 Ethidium bromide-stained agarose gel showing PCR products. Lane M , 100-bp DNA ladder (Promega); lane 1 , set1B gene product; lane 2 , set1A gene product; lane 3 , ial gene product; lane 4 , ipaH gene product; lane 5 , mPCR product. Initial attempts to amplify equally all the four genes in a single reaction using the reaction condition in monoplex PCR were not successful. A common practice in mPCRs involving any non-amplification of a required gene ('weak locus') is to increase the amount of primers of the gene at same time with a decrease of the amount of primers for all the loci that can be amplified, especially those with strong amplifications. Hence, the concentrations of primers for both ipaH (Shig) and set1B (ShET1B) were reduced to 0.3 μM each and the primers for both set1A (ShET1A) and ial (ial) genes were maintained at 0.5 μM each. Following optimization of the concentrations of Taq DNA polymerase (0.6 to 4U/25 μL), buffer strength (1.4 X to 2.4 X), dNTPs (140 to 220 μM) and annealing temperatures (49 to 59°C) (at a constant MgCl 2 concentration of 4 mM), a more uniform amplification of all the genes with no background noise was obtained (Fig. 1 lane 5) at a final buffer concentration of 1.8X, 1 U Taq DNA polymerase, 130 μM dNTP each and annealing temperature of 55°C. Prevalence of virulence genes in the Malaysian strains All the 110 strains of Shigella spp. tested showed the presence of ipaH (Table 2 ). Conversely, only 41% of the strains had both set1A and set1B genes, and ial gene. Almost all the Shigella strains tested positive for the tandem genes (87%) belonged to S. flexneri 2a serotype. Among the predominant strains of Shigella flexneri in Malaysia, ial was found in serotypes 4a, 6, 3a, 2a, 1a and 3c. All the four genes were present only in S. flexneri 2a and 3a. Table 2 Prevalence of the four virulence-associated genes in Malaysian Shigella spp. Serotype Total strains set1B (%) set1A (%) ial (%) ipaH (%) S. flexneri 1a 3 0 (0) 0 (0) 1 (33) 3 (100) 1b 3 0 (0) 0 (0) 0 (0) 3 (100) 2a 47 41 (87) 41 (87) 19 (40) 47 (100) 3a 18 3 (17) 3 (17) 12 (67) 18 (100) 3c 10 0 (0) 0 (0) 1 (10) 10 (100) 4a 1 1 (100) 1 (100) 1 (100) 1 (100) 6 1 0 (0) 0 (0) 1 (100) 1 (100) y 1 0 (0) 0 (0) 0 (0) 1 (100) S. sonnei 15 0 (0) 0 (0) 2 (13) 15 (100) S. dysenteriae 2 10 0 (0) 0 (0) 8 (80) 10 (100) S. boydii 6 1 0 (0) 0 (0) 0 (0) 1 (100) Total 110 45 45 45 110 Reproducibility Reproducibility for the detection of set1A, set1B, ial and ipaH genes assayed in the mPCR was 100%. None of the non- Shigella strains tested gave any amplification (data not shown). Sensitivity The mPCR assay was tested on 10-fold dilutions of an overnight culture of S. flexneri 2a. All the four virulence-associated genes were detected until 10 -3 dilutions (data not shown). This was equivalent to 2.45 × 10 5 lysate or a minimum of 490 cfu of shigellae per 25 -μLmPCR reaction. Faecal spiking and sensitivity An initial experiment using undiluted spiked faecal sample failed to give any PCR amplification (data not shown). When the faecal suspension was diluted and preincubated in BHI for 4 h, the mPCR assay was successful in detecting the presence of the four virulence genes at an average concentration of 5.0 × 10 4 colony-forming units (cfu) shigellae ml -1 or approximately 100 cfu per reaction mixture (Fig. 2 lane 8). Figure 2 Faecal-spiking and sensitivity result of mPCR. Lane M , 100-bp DNA ladder (Promega, USA); lane 1 , TH13/00 (positive control); lane 2 , unspiked faeces (negative control); lane 3 , undiluted spiked faeces; lane 4 , 10 -1 dilution; lane 5 , 10 -2 dilution; lane 6 , 10 -3 dilution; lane 7 , 10 -4 dilution; lane 8 , 10 -5 dilution; lane 9 , 10 -6 dilution; lane 10 , 10 -7 dilution; lane 11 , 10 -8 dilution; lane 12 , 10 -9 dilution; lane 13 , 10 -10 dilution; lane 14 , "water blank". Clinical specimens A preliminary study on the efficacy of the mPCR assay in the direct detection of the aforementioned Shigella virulence genes on faecal samples was tested on ten diarrhoeal patients. No mPCR product was detected although both the positive controls had amplifications. By conventional culture method, there was no growth of Shigella on the LB, MacConkey and SS agar plates. Discussion Numerous studies had been performed to detect virulence genes in Shigella by monoplex PCRs [ 8 , 17 , 18 ]. Studies involving the combination of chromosomal- and plasmid-encoded virulence genes in a single assay for Shigella detection, on the other hand, are scarce. Although the optimization of mPCR is more tedious and difficult to achieve than monoplexes, the ease of screening a large number of specimens, once the system is optimized, far outweighs the initial problems. The present mPCR system encompasses the presence of virulence genes found in the Shigella chromosome and on the large inv plasmid. Hence, it can determine if the pathogenesis of a particular strain is attributable to its chromosome or the plasmid, or if the strain is still invasive or otherwise, in a single reaction. Initially, the monoplex PCRs were carried out following reaction conditions as proposed by a previous report [ 17 ]. However, we could not reproduce their results and hence had to modify the PCR conditions. Our failure to reproduce identical results despite using similar reagent concentrations and amplification conditions maybe attributed to the different makes of PCR reagents and primers used. Broude et al . [ 19 ] had compared amplification efficiencies of two commercial Taq DNA polymerases and found that they displayed different specificity in PCR. Preferential amplification of one target sequence over another is a known phenomenon in mPCRs and it is usually overcome by increasing the amount of primers for the weaker amplification simultaneously with a decrease of primer concentrations for the stronger amplification. Buffer concentration may also affect mPCR amplifications despite it being seldom considered during monoplex optimization works [ 20 ]. Upon adjustment of primers and buffer concentrations, specific and consistent amplification of all the genes in the multiplex combination was achieved. Although other studies have demonstrated the presence of ial and ipaH in strains of enteroinvasive Escherichia coli (EIEC) [ 11 , 13 , 21 ], we had not applied the mPCR assay to EIEC strains. It is unfortunate that these strains were not available for our study as EIEC gives rise to similar illness as Shigellosis. Our study supported the observations of Noriega et al . [ 9 ] and Vargas et al . [ 17 ] in local Shigella strains. Their studies showed that both set1 A and set1 B were present exclusively in S. flexneri 2a. The complete correlation between the presence of both set1A and set1B showed that both genes are indeed found in tandem in the Shigella genome. In this study, almost all the Shigella strains positive for the presence of set1A and set1B (41/45 strains) belonged to S. flexneri 2a, thus confirming previous works that both genes are highly conserved in this particular serotype [ 14 ]. Both the prevalence of ial and ipaH were independent of the four different species of Shigella tested. Though both ial and ipaH are responsible for invasion-related processes and are found on the inv plasmid, the ial gene cluster resides near a region of the plasmid, which is a hot spot for spontaneous deletions [ 22 ]. This probably explains the lower prevalence of ial (45/110 strains) than ipaH (110/110 strains) in the Malaysian Shigella strains. Since invasiveness is a prerequisite for virulence in shigellae and since most of these virulence genes are located on the large plasmid, these strains would have possessed the plasmid when first isolated from patients. Due to storage/subculturing, the plasmid might have been lost together with the virulence-associated genes. By virtue of multiple copies being present on both the chromosome and the inv plasmid [ 23 ], ipaH seemed to be less compromised by plasmid loss and/or deletions. As the sole presence of ipa H is not indicative of the invasive phenotype, our mPCR design, which incorporated three other virulence genes, could determine the invasiveness of Shigella strains in epidemiological studies. Dilution of the faecal sample with BHI was performed to lower the levels of PCR inhibitors such as bilirubin, bile salts and heme in the faeces [ 16 ]. An additional step of preincubating the spiked faecal samples also helped to eliminate the natural inhibitors [ 24 ]. The short 4-h enrichment step would increase the total number of target sequences caused by more bacterial growth and the overall detection sensitivity of the assay. Although PCR cannot differentiate between dead and viable bacteria, enrichment helped to dilute the concentrations of dead bacteria, thus reducing the probability of detecting them by the subsequent mPCR assay. The sensitivity level achieved in the study was found to be comparable to other studies. Houng et al . [ 25 ] detected up to 7.4 × 10 4 cfu shigellae ml -1 by amplifying the IS 630 sequences in shigella spp.. Yavzori et al . [ 24 ] reported a detection level of 10 4 cfu shigellae per gram of faeces with the use of virF primers. Although it has been reported that ingestion as low as 100 shigellae resulted in clinical disease [ 26 ], the highest percentage of volunteers having diarrhoea were administered doses of at least 10 4 viable organisms. Thus, the average detection limit of mPCR described in this study (5.0 × 10 4 cfu/ml) is within the common infectious dose for shigellae . Results from the preliminary clinical screening were promising. Nevertheless, the consideration of other diarrhoeal pathogens being present in the clinical samples cannot be negated. More patient samples are warranted to thoroughly vet the robustness and applicability of the developed mPCR in clinical environments. One limitation of the present mPCR system is its inability to differentiate Shigella spp., unlike the multiplex reactions based on specific rfc genes developed by Houng et al . [ 25 ]. For future research, either set1A or set1B may be omitted from the multiplex system as both genes are shown to exist tandemly. rfc primers of different Shigella origins maybe incorporated to enable the discrimination of Shigella spp. as well as the identification of virulent strains in one assay. Conclusions We conclude that the mPCR system is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. The described assay is reproducible, sensitive, can be easily performed and is able to give a presumptive identification of the causal pathogen, which could be confirmed by culture techniques using selective media. An added advantage would be that EIEC, which gives a similar illness, might also be detected by this method, as EIEC also harbours ial and ipaH genes. Competing interests The author(s) declare that they have no competing interests. Authors' contributions SLLH carried out the experiments, data analysis and wrote the manuscript. RMY provided the bacterial strains. SDP contributed to the writing of the manuscript. TKL conceived and co-designed the study, provided input for writing and supervision of the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551607.xml |
522819 | Magnesium administration provokes motor unit survival, after sciatic nerve injury in neonatal rats | Background We examined the time course of the functional alterations in two types of muscles following sciatic nerve crush in neonatal rats and the neuroprotective effect of Mg 2+ . Methods The nerve crush was performed on the 2 nd postnatal day. MgSO 4 *7H 2 O was administered daily for two weeks. Animals were examined for the contractile properties and for the number of motor units of extensor digitorum longus and soleus muscles at three postnatal stages and adulthood. Four experimental groups were included in this study: i) controls, ii) axotomized rats, iii) magnesium treated controls and iv) axotomized and Mg 2+ -treated rats. Results Axotomy resulted in 20% MU survival in EDL and 50% in soleus. In contrast, magnesium treatment resulted in a significant motor unit survival (40% survival in EDL and 80% in soleus). The neuroprotective effects of Mg 2+ were evident immediately after the Mg 2+ -treatment. Immature EDL and soleus muscles were slow and fatigueable. Soleus gradually became fatigue resistant, whereas, after axotomy, soleus remained fatigueable up to adulthood. EDL gradually became fastcontracting. Tetanic contraction in axotomized EDL was just 3,3% of the control side, compared to 15,2% in Mg 2+ -treated adult rats. The same parameter for axotomized soleus was 12% compared to 97% in Mg 2+ -treated adult rats. Conclusions These results demonstrate that motoneuron death occurs mostly within two weeks of axotomy. Magnesium administration rescues motoneurons and increases the number of motor units surviving into adulthood. Fast and slow muscles respond differently to axotomy and to subsequent Mg 2+ treatment in vivo. | Background In the rat the first 3 weeks of life constitute a critical period of neuromuscular plasticity. Contractile properties of muscles are not inherent, but are determined by the motor nerve, that supplies the muscle. This was shown by experiments of crossinnervation between fast and slow muscles [ 1 ]. Axotomy of peripheral nerves in neonatal rats, leads to loss of the bigger immature motoneurons through an excitotoxic process, with bigger neurons firing at a greater frequency. Consequently there is loss of the fast-contracting muscle fibers being innervated by bigger nerve cells [ 2 ]. There is much evidence that overactivation of glutamate receptors plays a significant role in this process (glutamate excitotoxicity) [ 3 ]. Glutamate is a major neurotransmitter in the CNS. Ionotropic receptors of glutamate (NMDA and AMPA / kainate) have been identified throughout the brain and spinal cord in several species of animals, including humans [ 4 ]. Their activation leads to Ca 2+ influx into the cell and subsequent activation of a cell death cascade (activation of proteases, lipases and other enzymes leading to cell lysis). Following neonatal sciatic nerve injury, the surviving motoneurons, take at least 8 days to grow back to the hind limb muscles, whereas most of the motoneurons that die, do so by apoptosis within the first two days [ 5 ]. However, it has been shown by previous studies [ 6 , 7 ] that motoneurons are highly vulnerable to excitotoxic cell death, only during the first five days of postnatal life. Sciatic nerve injury by axotomy in neonatal rats has been shown to result in significant reduction in the number of surviving motoneurons in the ventral horn of the lumbar segments. Developing motoneurons become resistant to axon injury when their axon terminals are converted from growing into transmitting structures and form stable connections with the muscle fibers they innervate [ 8 ]. Target deprivation from muscle after the 5 th postnatal day (P5) does not cause significant motoneuron loss, since damaged axons reinnervate muscle fibers successfully [ 6 , 7 , 9 ]. In this study we examine the possible neuroprotective effects of systemic administration of magnesium ions, after axotomy in neonatal rats on the 2 nd postnatal day. It could be assumed that in our model, magnesium ions penetrate the blood brain barrier, as magnesium has been shown to concentrate in the cerebrospinal fluid after intraperitoneal injection and increase the electrical threshold required to control seizures, in a rat model, by other investigators [ 10 ]. We also investigated the time course of motor unit loss and the alterations in the contractile properties of a fast- (EDL) and slow-contracting (soleus) muscle. Methods Experimental procedures Wistar Albino rats of both sexes were used in these experiments. All efforts were made in order to minimize the number of animals used and their suffering. The research project complies with the guidelines for animal use, established by the American Physiological Society and was approved by the local ethical committee in accordance with EEC Council Directive 86/609. The day of birth was taken as P0 (zero). At the 2 nd postnatal day the sciatic nerve of the left hindlimb was crushed, in order to perform axotomy. Four experimental groups were included in this study: i) control, ii) rats whose sciatic nerve was crushed (axotomy), iii) magnesium treated controls and iv) rats whose sciatic nerve was crushed and were magnesium treated. The rats were examined for the contractile properties and the motor unit number of two hindlimb muscles (EDL and soleus) at several stages of postnatal development. Tension recordings were held in both hindlimbs (operated and control) in groups of Mg 2+ -treated and non-treated rats at: a) Postnatal day 14 (P14), b) Postnatal day 21 (P21), c) Postnatal day 28 (P28) and d) Adult rats (older than 2 months). In our study six successfully tested Mg 2+ -treated and six non-treated rats of each age-group (48 rats) were included. Nerve crush The animals were anesthetized with ether at the 2 nd postnatal day and were operated under sterile conditions. Surgery was performed under an operating stereoscope (10× magnification). The sciatic nerve was identified and crushed at the mid-thigh, just proximal to its division to the tibial and common peroneal nerves. Care was taken not to damage the blood supply to the surrounding tissues. Crush was performed using a fine forceps for 30 seconds. Then the nerve was examined to ensure that the epineural sheath was intact but translucent (axotomy). The wound was closed with fine sutures. All surgical procedures were carried out by the same surgeon. Three hours after wound suturing the neonates were placed back with their mothers. Specific tests were performed daily in order to confirm the efficacy of axotomy: a) The plantar- and the dorsi-flexion reflexes were checked: The examiner, placing his index finger on the animals foot, forces it to plantar- and dorsi-flex the ankle. The animal reflects actively, doing the opposite movement, if able to dorsi- and plantar-flex, respectively. No reactive movements are evident after successful denervation. b) Animals were suspended by their tail and the inability of normal movement of the left hindlimb, indicating successful nerve axotomy, was assessed for the following [ 11 ]: _ Hip and knee in extension _ Ankle in the operated side in plantar flexion _ Adduction of the whole limb _ Weakness of digit extension in operated side The first behavioral signs of reinnervation after axotomy, should be evident at about 10–12 days after injury, according to other investigators [ 12 ]. Only those animals, whose successful axotomy was verified, were included in our study. Magnesium administration The rats of some litters were treated daily with magnesium ions. Mg 2+ was injected subcutaneously as a solution of MgSO 4 7H 2 O (0,05 ml of 1 M solution / 10 g body weight). This was the highest tolerable dose. Mortality in higher doses exceeded 80%. The treatment continued until the rats were 14 days old. Control groups were injected daily with the same volume of normal saline (0,05 ml / 10 g body weight). In Mg 2+ treated animals weight gain slowed down during the first month of their life. Treated and control adult rats, however, did not show statistically significant differences in their body weight. Axotomized rats, presented with motion deficit on the denervated limb, as described above, for about 2 weeks. After Mg 2+ treatment normal kinetic behaviour and reflexes returned on the 7 th to 9 th postnatal day. Tension recording Animals were anesthetized with Chloral Hydrate (4,5% 1 ml / 100 g of body weight). The EDL and soleus muscles of both the axotomized and the contralateral control leg were prepared. The distal tendons were dissected free and attached to a strain gauge transducer (Dynamometer UFI, Devices) by a short silk suture and the exposed parts of the muscles were kept moist with warm (37°C) Krebs solution (NaCl 118,08 mM, NaHCO 3 25 mM, glucose 5,55 mM and CaCl 2 1,89 mM) [ 13 ]. The sciatic nerve was exposed. In order to isolate soleus muscle contraction and to avoid summation of concomitant gastrocnemius muscle contraction, after stimulating the sciatic nerve we used to cut the branches innervating the gastrocnemius and plantaris longus muscles. The leg was held rigidly in a position of 90° flexion of the knee and ankle joints, by two pins, inserted in the femoral condyles and the calcaneus, respectively. The tendons of EDL and soleus muscles were connected to a strain gauge and the tension elicited by sciatic nerve stimulation (Digitimer DS9A stimulator) was displayed on the screen of an oscilloscope (Fluke PM 3380 A). Muscle length was adjusted so as to produce maximal single twitch tension (optimal length), through a micromanipulator allowing motion on the 3 axes (Prior, England). Then stimulus intensity was adjusted in order to elicit maximal tension, using supramaximal (3–9 volts) square pulses of 0,5 msec duration. The signal from the transducer was amplified by a DC transducer amplifier (Neurolog NL 107). Time To Peak (TTP) and Half Relaxation Time (HRT) of the Single Twitch recording were measured. Tetanic contractions were then elicited by stimulating the nerve at 10, 20, 40, 80 and 100 Hz. The duration of stimulus was 250 msec. All devices during the tension-recording procedure were controlled by a pulse programmer (Digitimer D4030). The number of motor units (MU) was estimated by the incremental method (the number of different single twitch tensions produced by stepwise increments of stimulus intensity). The fatigueability of the muscles was tested by stimulating them at 40 Hz for 250 msec every second. The decrease in tension at 3 minutes of such stimulation was measured and the % Fatigue Index (FI) was calculated: F.I. = (Initial Tension - Tension at 3 min) / Initial Tension. After tension recordings were completed, muscles were excised and weighed. The animals were killed by cervical dislocation. Statistical analysis Stiatical analysis of the results was performed using SPSS 10.0 software for Windows. Nonparametric tests (Mann – Whitney for two independent variables and Kruskal – Wallis for more than two independent variables) were used in order to compare data, of different groups. Criterion of statistical significance was set at p < 0.05. Results and discussion Number of motor units The number of motor units was estimated by the stepwise increments of tension, created by stimuli of different intensity. Our findings are in consistency with results of previous work by other authors [ 14 ], such as by our previous experience [ 7 ]: The control EDL muscle contains approximately 40 motor units, whereas soleus of 30 motor units, independently of animal age (Table 1 , Figures 1a and 1b ). Treatment with Mg 2+ does not affect the number of motor units of control muscles. Axotomy at P2 results in statistically significant (p < 0.05) motor unit loss in both EDL and soleus in all age groups. This means that motoneuron death is already established at P 14 . Treatment with Mg 2+ results in a statistically significant difference in the survival of motor units compared to non-treated axotomized rats. It is obvious that the neuroprotective effect of Mg 2+ is already established at P14 immediately after the period of treatment (Table 1 , 2 , 3 and Figures 1a and 1b ). Figure 2 , images the different single twitch tension recordings, elicite, in an EDL muscle on the left leg (side of nerve injury), after stimulation of the left sciatic nerve with electrical stimuli of incrementally increasing intensity. In this case, the axotomized EDL muscle consisted of six motor units, that survived axotomy. Tension development Axotomy affects tension development by the muscle (Tables 4 and 5 ). In adults rats single twitch of EDL is 4.63 ± 0.78% of the control muscle, whereas this of soleus is 16.80 ± 3.03% (Tables 2 and 3 ). Maximal Tetanic Tension is being developed by stimulation at 100 Hz. Maximal Tetanic Tension in adult rats is only 3.31 ± 0.30% of the control side, whereas after Mg 2+ treatment it is 15.16 ± 0.89% respectively. The excessive discrepancy of force outcome ability by both muscles between P28 and adulthood is also noticeable: At P28 respective values are: 18.94 ± 4.16 vs. 40.14 ± 19.34%. At P21 respective values are: 23.35 ± 16.03% vs. 64.22 ± 43.02% (Tables 2 and 3 ). The absolute values of Tetanic Tension at 100 Hz as listed in Tables 4 and 5 , are statistically significant different, between the operated (left) and the control (right) side. At P14, on the other hand, a tension development deficit does not develop after axotomy. Maximal Tetanic Tension of soleus is also being reduced after axotomy: 12.44 ± 0.97% of the control side in adult rats. Respective values in the other age groups are: 67.39 ± 39.21% at P28, 79 ± 14.34% at P21 and 81.96 ± 13.56% at P14 (Tables 2 and 3 ). This marked reduction of tension developing ability of both EDL and soleus, in adult rats, is established after the first month of life. An obvious explanation is the excessive muscle atrophy that occurs gradually as the animal grows up. Mg 2+ treatment, which results in inhibition of muscle atrophy, has been shown to promote motor units survival, and causes the axotomized soleus to remain as strong as the control muscle. Maximal Tetanic Tension of axotomized compared to control side is 97 ± 11.33% in adult rats, 91.45 ± 15.09% at P28, 77.01 ± 25.63% at P21 and 92.12 ± 11.15% at P14 (Tables 2 and 3 ). Figure 2 shows representative recordings of twitch and titanic tensions, as well as fatigue indexes of EDL and soleus muscles; tables 2 , 3 , 4 and 5 summarize all the above results. Contraction velocity EDL is normally a fast contracting muscle in adult rats, whereas soleus is a slow one. Immature (P 14 ) muscles, however, are not yet differentiated into fast- or slow-contracting. In adult rats TTP and HRT are respectively a) 36 ± 4.4 msec and 28 ± 2.28 msec in the EDL and b) 56.2 ± 3.37 msec and 59.5 ± 3.78 in the soleus (Table 6 ). At P14 in the EDL, TTP is 56 ± 8.16 msec and HRT is 59 ± 4.34 msec, whereas the respective values for soleus are 74 ± 8.29 msec and 66 ± 8 msec. Axotomy gradually converts EDL into a slow-contracting muscle: TTP is 56 ± 4.97 at P14, 49 ± 10.18 at P21, 43 ± 5.48 at P28 and 77 ± 7.89 in adult rats (Table 6 ). These values are significantly different to the ones obtained from control muscles. Soleus on the other hand, remains slowcontracting following axotomy, in all age groups. Axotomy does not alter soleus contraction velocity (no statistically significant difference between values). Mg 2+ administration did not affect contraction and relaxation velocity of control muscles (no statistically significant difference). It caused axotomized EDL to become fast-contracting in adult rats (TTP: 38 ± 7.53 and HRT: 43 ± 4.13), as it is predicted for control muscles, whereas soleus' contractility was not affected. Fatigueability Soleus is a fatigue resistant muscle in adult rats, whereas EDL is not. However, both immature EDL and soleus muscles show properties of fatigueable muscles. EDL (control or operated) at P14 is not fatigue resistant, irrespective of Mg 2+ administration, with a fatigue index of about 65% (Table 6 ). During normal development EDL remains fatigable to adulthood, with Mg 2+ treatment not affecting this state. Axotomy however, causes EDL to become fatigue resistant in adult rats (F.I = 15.6). Values of operated side compared to those of control muscles, are statistically significantly different even at P21. Mg 2+ administration after axotomy does not induce conversion of EDL to a fatigue resistant muscle. Soleus While soleus is not fatigue resistant at P14 (F.I.= 55.6%), it gradually becomes fatigue resistant during normal development (Table 6 ). Axotomy on the other hand, results in soleus becoming less fatigue resistant in adult rats (F.I. = 34.7%, statistically significant different than control muscles). However, if axotomy is combined with Mg 2+ treatment, the development of soleus into a fatigue resistant muscle is not hindered. Muscle weight Muscle weight on the operated side is statistically significantly reduced compared to the contralateral control side. It is noticeable, however, that there is a marked reduction in muscle weight from P28 to adulthood both in the EDL and the soleus. The reduction in muscle weight of axotomized EDL was already established at P14, whereas in soleus there was a statistically significant difference only after P28 (Tables 4 and 5 ). Mg 2+ administration provokes muscle weight increase on the operated side. In soleus, values are almost equal to those of control muscles, whereas in EDL the weight gain is less dramatic (Tables 4 and 5 ). Our results support findings of previous studies by other workers, concerning the alterations in muscles and motor units after axotomy of peripheral nerves in neonatal rats. In the present study we concentrate on the time course of these alterations. We also focus on the influence of the in vivo administration of magnesium sulphate on motor unit survival and consequently on enhancing force outcome and muscle weight improvement. Administration of an NMDA or an AMPA receptor antagonist within this critical initial period of development, is thought to reverse the neurotoxic effects of axotomy and results in increased survival of motoneurons. Dizoscilpine malate (MK-801), an NMDA antagonist, has been used in animal models in vivo with success, in order to prevent motoneuron death after axotomy [ 15 , 16 ]. However, it was badly tolerated by rats, due to side effects (high mortality). Magnesium is a non-competitive, voltage dependent, NMDA-receptor antagonist, acting by coupling with the specific Mg 2+ site within the pore of the ion channel [ 17 - 19 ]. Its similarity of action compared to MK-801 has been shown in two experimental models of neuropathic pain [ 20 ]. Moreover, axotomy in early postnatal period does not only reduce the number of surviving motoneurons and motor units, but also provokes changes in the contractile properties of limb muscles [ 21 ]. Immature muscle fibers have not gained yet the characteristics of fast- or slow- contracting type, since not all subtypes of the contractile proteins and enzymes have yet been formed. Extensor Digitorum Longus (EDL), for example, is normally a fast – contracting and fatigable muscle in the adult rat. If axotomy is performed in neonates, EDL becomes slow and fatigue resistant. As other investigators have shown before [ 22 ], changes in the contractile properties of immature muscles, during normal development, go on for 30 days after birth, although establishment of mononeuronal innervation of muscle fibres is already fulfilled at P15 and the number of motoneurons innervating a specific muscle is constant after P0. It has been shown that the number of motor units remains constant after birth [ 14 , 23 ]. This is consistent with our results: The EDL consisted of 40 and the soleus of 30 motor units in all age groups. It should be mentioned that soleus contractile properties are not significantly altered throughout early postnatal life. Soleus is a slow, non-fatigue resistant muscle at P14 that progressively becomes fatigue resistant. The process of soleus' development into a fatigue resistance muscle is stopped, after sciatic nerve axotomy. Axotomized soleus becomes less fatigue resistant in adult rats, compared to control muscles. However the process of muscle necrosis, as proposed by other authors, could contribute to this result, as well, rather than the loss of motor units alone [ 25 ]. Denervated soleus muscle at birth is less fatigue resistant than control muscles in adult rats. This state is already established at P28, with this process progressing further on to adulthood. As mentioned already, it is noticeable that force outcome even by this 'conservative' muscle is largely affected by axotomy as the animal grows up, after the first month of life. Our data show that even when no marked reduction in the number of motor units occurs, muscle weight does not improve from P28 to adulthood neither does single twitch or maximal tetanic tension. The same phenomenon appears in the EDL as well. The discrepancy seen during the P28 to adulthood interval, both in EDL and soleus, between the reduction in the number of motor units on the one hand, and force outcome (single twitch and tetanic twitch tension) and muscle weight after axotomy on the other, can be explained as a consequence of marked muscle atrophy and necrosis. As shown by other workers [ 24 ], all immature muscle fibers denervated at birth fail to become reinnervate, and the few reinnervated muscle fibers may be overloaded, hypertrophied and eventually necrotized. The final level of tension achieved by the denervated muscle, represents the equilibrium, between decrease in force due to atrophy and necrosis due to regeneration [ 25 ]. It has to be considered that nerve injury during early post-natal life, causes permanent changes in the muscles that are not caused by motoneuron death. Both immature EDL and soleus muscles are slow contracting at P14. During normal development EDL gradually converts into fast contracting, remaining not fatigue resistant throughout life. We found that EDL has already gained characteristics of a fast muscle at P21. Axotomy causing death of the bigger motoneurons, thus destroying the large motor units, converts the muscle into slow-contracting. It was also shown that increase in speed of both EDL and soleus was much reduced after denervation [ 26 ]. Our data show that EDL was affected by axotomy much more than soleus. If connection between muscle and nerve is disrupted, as is the case after axotomy, fastcontracting muscles are mostly impaired [ 12 , 21 , 27 ]. It is known that the overall poorer recovery of immature fast muscles after denervation seems to be due to preferential loss of fibers from fast motor units during reinnervation [ 28 , 21 , 29 ]. Histological findings [ 27 ] and studies on the isometric tension recordings by other workers [ 21 ], confirm our findings. However, there is one study [ 30 ], suggesting that soleus is the muscle predominately affected after nerve injury. Previous studies [ 6 ] have shown that sciatic nerve crush performed at 5–6 days after birth resulted in a 50% reduction in maximal tetanic tension of the EDL two months after injury, with the muscle becoming more fatigue resistant. Respective values for soleus, however, remained almost equal to those of control muscles. When nerve crush was performed at 3–5 days of life [ 7 ], single twitch tension values of the operated side were 36% of those observed for the control side. Respective value for maximal tetanic tension is 50% and for muscle weight is 61%. It is noticeable when comparing those data with our present results, that motoneurons at P2 are much more vulnerable to the excitotoxic effects of axotomy. Axotomy has also been shown to convert EDL into slow-contracting [ 7 , 27 ]. On the other hand repeated injury to the sciatic nerve (at 5 and again at 11 days), has been shown to cause motoneuron death, mostly to the soleus, compared to the tibialis anterior & EDL motoneuron pool, in the spinal cord ventral horn [ 9 ]. Magnesium administration by other workers [ 31 ] has been shown to cause only a slight improvement in motoneuron survival after nerve crush at birth. However, the same authors found that daily in vivo administration of magnesium sulphate accompanied by NMDA, in axotomized rats at P5, rescues motoneurons destined to die. Our results are strongly suggestive that daily systemic treatment with magnesium sulphate, in order to keep sufficient blood concentrations of magnesium ions, results in increased motor unit survival. Conclusions Our findings strongly support the findings of previous work [ 31 ], that has shown magnesium in vivo administration to rescue sciatic motoneurons from cell death, after axotomy. Local application of magnesium ions to the muscle, by implants affecting achetylcholine release in the neuromuscular junction [ 32 ], is suggested to reduce the number of surviving motoneurons by some other mechanism than blocking glutamate receptors. In our study however magnesium ions were not applied directly on the neuromuscular junction. It could be assumed that our results represent the resultant of magnesium ions actions on the nerve and the muscle. Magnesium administration did not cause any statistically significant influence on the contractile properties of the control muscles in the right unoperated leg, in any age group. Neuroprotection by magnesium reversed the effects of excitotoxicity, predominately in the fast-contracting EDL. In conclusion, our results show that motoneuron death occurs mostly within two weeks of axotomy, while systemic Mg 2+ administration rescues motoneurons and increases the number of motor units surviving into adulthood. Furthermore, fast and slow muscles respond differently to axotomy, as well as, to subsequent in vivo treatment with Mg 2+ . Competing interests The authors declare that they have no competing interests. List of abbreviations AMPA: a-amino-3-hydro-5-methyl-4-isoxazolo-propionic acid CNS: Central Nervous System EDL: Extensor Digitorum Longus muscle FI: Fatigue Index HRT: Half Relaxation Time Mg: Magnesium MU: Motor Units NMDA: N-methyl-D-aspartate P: Postnatal SD: Standard Deviation TTP: Time to Peak Authors' contributions NG carried out the experiments, participated in the sequence alignment as well as in the design of the study and drafted the manuscript. AH participated in the experiments and performed the statistical analysis DK participated in the experiments MA conceived of the study, and participated in its design and coordination Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522819.xml |
549712 | The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal | Background Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr) on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo , we inactivated the Ptdsr gene in the mouse. Results Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr -/- mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr -deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. Conclusion Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance. | Background Programmed cell death, or apoptosis, is required for the normal development of almost all multicellular organisms and is a physiological mechanism for controlling cell number; as a result, structures that are no longer needed are deleted during development and abnormal cells are eliminated [ 1 , 2 ]. Most of the cells produced during mammalian embryonic development undergo physiological cell death before the end of the perinatal period [ 3 ]. Apoptotic cells are removed rapidly and efficiently as intact cells or apoptotic bodies by professional phagocytes or by neighboring cells. This highly regulated process prevents the release of potentially noxious or immunogenic intracellular materials and constitutes the fate of most dying cells throughout the lifespan of an organism [ 4 , 5 ]. Phagocytosis of apoptotic cells is very distinct from other engulfment processes that result, for example, in the clearance of microorganisms, because engulfment of apoptotic cells triggers the secretion of potent anti-inflammatory and immunosuppressive mediators, whereas pathogen recognition causes the release of pro-inflammatory signals [ 6 ]. Almost all cell types can recognize, respond to, and ingest apoptotic cells by using specific sets of phagocytic receptors that bind to specific ligands on apoptotic cells. Detailed genetic studies in Drosophila and Caenorhabditis elegans have recently yielded evidence that basic phagocytic mechanisms and pathways for the recognition and engulfment of apoptotic cells are highly conserved throughout phylogeny [ 7 , 8 ]. In vertebrates, a number of receptors have been identified that can mediate phagocytosis of apoptotic cells. These include, for example, scavenger receptors and pattern recognition receptors such as CD36, SR-A and CD14, integrins such as the vitronectin receptor α v β 3 , and members of the collectin family and their receptors CD91 and calreticulin [ 9 - 13 ]. The individual roles of these molecules in binding, phagocytosis or transduction of anti-inflammatory signals upon apoptotic cell recognition have not been well defined, however [ 5 , 6 , 14 ]. The importance of efficient mechanisms for apoptotic cell clearance in vivo is supported by the observation that autoimmune responses can be provoked in mice when key molecules for apoptotic cell recognition and uptake are missing. This has been reported for knockout mice lacking the complement protein C1q [ 15 ], for mice with a mutation in the tyrosine kinase receptor gene Mer [ 16 ] and, more recently, in mice lacking transglutaminase 2 or milk fat globule epidermal growth factor 8 (MFG-E8) [ 17 , 18 ]. The exposure of the phospholipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane of apoptotic cells has been described as one of the hallmarks of the induction of apoptosis and is considered to be one of the most important signals required for apoptotic cell recognition and removal [ 19 ]. A number of cell-surface and bridging molecules can interact with exposed PS on apoptotic cells. These include the serum proteins β2-glycoprotein 1 and protein S [ 20 , 21 ], the growth-arrest-specific gene product GAS-6 [ 22 ], complement activation products [ 23 ], the milk fat globule protein MFG-E8 [ 24 ], and annexin I [ 25 ]. In most cases the receptors on phagocytes that recognize these PS-bridging molecules have not been defined, but it has been reported that GAS-6 is a ligand for the tyrosine kinase receptor Mer and that MFG-E8 can bind to the vitronectin receptor α v β 3 [ 16 , 24 ]. Other molecules that bind PS with varying specificity are the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the scavenger receptors CD36 and CD68 (for review see [ 5 ] and references therein). The best-characterized molecule so far that binds PS in a stereo-specific manner is the phosphatidylserine receptor (Ptdsr) [ 26 ]. In vitro , it has been shown that the Ptdsr can mediate the uptake of apoptotic cells and that such Ptdsr-mediated phagocytosis can be inhibited through addition of PS liposomes, the PS-binding molecule annexin V or an anti-Ptdsr antibody [ 26 ]. Moreover, the binding of Ptdsr to PS on apoptotic cells has been reported to be important for the release of anti-inflammatory mediators, including transforming growth factor-β1 (TGF-β1), platelet-activating factor (PAF), and prostaglandin E2 [ 26 , 27 ]. These data supported the hypothesis that Ptdsr fulfils a role as a crucial signaling switch after the engagement of macrophages with apoptotic cells and is thereby fundamental for preventing local immune responses to apoptotic cells before their clearance [ 28 ]. Very recently, Ptdsr has been found in the cell nucleus. Its nuclear localization is mediated by five independent nuclear localization signals, each of which alone is capable of targeting Ptdsr to the cell nucleus [ 29 ]. Moreover, an additional study performed recently in Hydra showed an exclusively nuclear localization for the Ptdsr protein [ 30 ]. Most interestingly, the nuclear localization of Ptdsr in Hydra epithelial cells did not change upon phagocytosis of apoptotic cells. These reports challenge the original hypothesis, according to which Ptdsr is an exclusively transmembrane receptor for apoptotic cell recognition and anti-inflammatory signaling. To examine further the role of Ptdsr in vivo , we performed gene-expression and gene-targeting studies in mice. A perinatally lethal phenotype was observed in Ptdsr- knockout mice, and Ptdsr -deficient embryos displayed multiple defects in tissue and organ differentiation. While this work was in progress, both Li et al. [ 31 ] and Kunisaki et al. [ 32 ] also reported the generation and phenotypic characterization of Ptdsr -knockout mice. Of note, although some of their results were confirmed in our study, we found a fundamentally different phenotype with regard to clearance of apoptotic cells. Moreover, our study revealed marked and unexpected findings in Ptdsr -deficient mice that are not related to apoptosis. Results Generation of Ptdsr -deficient mice To investigate in vivo the functions of the phosphatidylserine receptor Ptdsr, we generated a null allele in the mouse by gene targeting (Figure 1a,1b,1c ). In contrast to previously described Ptdsr- knockout mice [ 31 , 32 ], we used Bruce4 embryonic stem (ES) cells for gene targeting [ 33 ], thus generating a Ptdsr -null allele in a pure, isogenic C57BL/6J genetic background. The newly established knockout mouse line was named Ptdsr tm1Gbf (hereafter referred to as Ptdsr -/- ). Heterozygous Ptdsr +/- mice were viable and fertile and showed no obvious abnormalities. Ptdsr +/- mice were intercrossed to generate homozygous Ptdsr -deficient mice. The absence of Ptdsr expression in Ptdsr -/- embryos was confirmed by RT-PCR (data not shown), and by northern and western blotting analyses (Figure 1d,e ). Interbreeding of heterozygous mice showed that the mutation was lethal, since homozygous mutants were not detected in over 100 analyzed litters at weaning. To determine the stages of embryonic development affected by the Ptdsr tm1Gbf mutation, timed breedings were followed by PCR genotyping (Figure 1c ) of embryos. We recovered fewer than the expected number of homozygous embryos from intercrosses of Ptdsr +/- mice. From a total of 1,031 embryos analyzed between gestational day (E) 9.5 and E18.5, 198 (19.2%) Ptdsr -deficient homozygous embryos were harvested, indicating that the introduced mutation is associated with a low rate of embryonic lethality in utero. From E9.5 to E12.5, Ptdsr -/- embryos were viable and of normal size. At E13.5 and thereafter, however, most Ptdsr -/- embryos showed morphological abnormalities (Table 1 ). All homozygous embryos harvested were growth-retarded from E13.5 onwards, had a pale appearance, and displayed multiple developmental dysmorphologies. These included various head and craniofacial malformations, such as exencephaly, cleft palate and abnormal head shape (Figure 1f,g ). Gross inspection revealed that eye development was severely affected in 14.1% of homozygous embryos. The affected animals displayed a complete unilateral or bilateral absence of the eyes (Table 1 ) that was never detected in Ptdsr +/+ or Ptdsr +/- littermates. Furthermore, homozygous embryos harvested between E12.5 and E15.5 had subcutaneous edema (Figure 1f,g ). Because we were able to recover Ptdsr -/- embryos until E18.5, we investigated whether Ptdsr -knockout mice could be born alive. Careful observation of timed matings allowed us to recover Ptdsr -/- neonates, but homozygous pups died during delivery or within minutes after birth. Ptdsr -deficient neonates were also growth-retarded, had a pale appearance and displayed various malformations. These included cleft palate, abnormal head shape, absence of eyes and edematous skin (Figure 1h ). Thus, deletion of the Ptdsr gene resulted in perinatal lethality with variable severity and penetrance of phenotypes. Expression of Ptdsr during embryogenesis and in adult tissues The observed perinatal lethality indicates that Ptdsr plays an important role during development. Analysis by RT-PCR (data not shown) showed that Ptdsr is expressed early in development, because we were able to detect Ptdsr transcripts in ES cells and embryos at all developmental stages. To analyze in more detail the temporal and spatial expression patterns of Ptdsr , and to correlate expression patterns with observed pathological malformations, we made use of a Ptdsr-β-geo gene-trap reporter mouse line generated from a Ptdsr gene-trap ES cell clone. This line has an insertion of β-galactosidase in the 3' region of the gene (Figure 2a ). We first examined Ptdsr expression by X-Gal staining in heterozygous embryos staged from E9.5 to E12.5. These developmental stages were chosen so as to investigate Ptdsr expression in affected organs prior to the onset of pathological malformations in Ptdsr -/- embryos. At E9.5 we found Ptdsr expression in the developing neural tube, somites, heart, gut and branchial arches (Figure 2b ). At E10.5, Ptdsr expression remained high in the developing nervous system, with most intense staining in the forebrain, hindbrain and neural tube. At this stage of embryogenesis, high levels of Ptdsr expression could also be detected in the developing limb buds and eyes (Figure 2b ). Ptdsr expression was altered at E12.5, with most intensive β-galactosidase staining in the eyes, developing condensations of the limb buds, neural tube and brain (Figure 2b ). Transverse sections of X-Gal-stained embryos at E12.5 showed an asymmetric expression pattern in the neural tube with intense staining of the central mantle layer but no expression in the dorsal part of the neural tube (for example, the roof plate; Figure 2c ). Expression in dorsal root ganglia lateral to the neural tube and in the somites was observed; Ptdsr was expressed throughout the somite structure (myotome, dermatome and sclerotome; Figure 2d ). Expression boundaries between somites were evident, with no expression in the segmental interzones, which correspond to the prospective intervertebral discs (Figure 2d ). Transverse sections of the developing eye at E12.5 revealed strong Ptdsr expression in the inner layer of the neural cup, which will later develop into the neural retina. Furthermore, Ptdsr expression was detected in the primary lens fiber cells of the developing lens (Figure 2e ). We carefully investigated whether Ptdsr is expressed from E10.5 to E12.5 in the developing kidney and lungs, but no expression could be detected indicating that Ptdsr expression is required only at later stages in the development of these organs (see below). Hybridization of a multiple-tissue northern blot revealed a single transcript of about 1.8 kb in almost every tissue analyzed in adult mice (Figure 2f ). The most prominent expression was observed in testis, thymus, kidney, liver and skin, with moderate to low expression in lung, small intestine, spleen, stomach and skeletal muscle. Thus, Ptdsr is ubiquitously expressed throughout embryogenesis and in adult tissues, although at different levels. Ptdsr is required for normal tissue and organ differentiation We next examined the role of Ptdsr in organ development. Serial histological sections of Ptdsr -/- and control embryos were taken to perform a detailed morphological analysis of all organ systems during development. A significant delay in organ and tissue differentiation was observed at E16.5 in lungs, kidneys and intestine. Lungs of control littermates were properly developed with expanding alveoli (Figure 3a ). Terminal bronchi and bronchioles were already well developed, and terminally differentiated epithelial cells with cilia on the luminal cell surface were present. In contrast, almost no alveoli or bronchioles were present in Ptdsr -/- lungs, indicating a delay or arrest in lung sacculation and expansion. Instead, we observed an abundance of mesenchyme that appeared highly immature (Figure 3g ). A similar delay in tissue differentiation of Ptdsr -/- embryos was found in the kidneys (Figure 3h ). Kidneys from Ptdsr +/+ embryos were well developed at E16.5, showing terminally differentiated glomeruli with Bowman's capsule and collecting tubules lined with cuboidal epithelial cells (Figure 3b ). In contrast, Ptdsr -deficient kidneys had only primitive glomeruli at E16.5, and collecting tubules were less well-developed. Instead, a large amount of undifferentiated mesenchyme was present in Ptdsr -/- kidneys (Figure 3h ). A delay in tissue differentiation was also found in the intestine at this stage of development. Ptdsr -/- embryos displayed improperly developed villi and an underdeveloped or absent submucosa (Figure 3i ). In wild-type embryos (Figure 3c ), intestinal cellular differentiation was already highly organized, with intramural ganglion cells between the external and internal muscular layers. Such neuronal cells were absent from the intestine of Ptdsr -/- embryos (Figure 3i ), however. Some Ptdsr -/- mice (4.5 %) also displayed extensive brain malformations that resulted in externally visible head abnormalities, with occasional ectopic tissue outside the skull or exencephaly (Figure 1f,h ). Histological analysis revealed an extensive hyperplasia of brain tissue with herniation of brain tissue either through the skull-cap or through the ventral skull (Figure 3d,j ). In the most severe cases, expansion of brain tissue in mutant mice resulted in further perturbations of cortical structures (Figure 3d,j ). Of note, a similar brain phenotype was observed in the Ptdsr -deficient mouse line generated by Li and colleagues [ 31 ]. In contrast to the study of Li et al. [ 31 ], however, we found almost normally developed lungs at birth. Ptdsr -/- lungs showed, in comparison to wild-type, only a slight delay in maturation and were fully ventilated in neonates in most cases (Figure 3e,k ). This demonstrates that Ptdsr -deficient mice can overcome the delay in embryonic lung differentiation and display normal lung morphology at birth. Thus, it would appear highly unlikely that Ptdsr -/- mice die from respiratory failure. Consistent with the observations of Kunisaki and colleagues [ 32 ], we found severely blocked erythropoietic differentiation at an early erythroblast stage in the liver (Figure 3f,3l ), suggesting an explanation for the grossly anemic appearance that we observed in our Ptdsr -/- mice. Loss of Ptdsr activity is associated with defects in ocular development and can lead to formation of ectopic eye structures By gross morphology we could differentiate two classes of Ptdsr mutants: those that appeared normal with both eyes present (Figure 4 ) and those that were severely affected and displayed uni- or bilateral anophthalmia (Figure 5 ). Analysis of normal or mildly affected embryos revealed no differences between mutant and wild-type embryos in the differentiation of the developing eye until E16.5. In both genotypes, inner and outer layers of the retina displayed a comparable differentiation status, as shown, for example, at E12.5 (Figure 4a,e ). At day E16.5, however, retinal layers in Ptdsr -/- embryos were much thinner than in wild-type embryos, contained fewer cells and were greatly reduced in size (Figure 4b,f ). Comparison of the retinal structures of Ptdsr +/+ and Ptdsr -/- embryos revealed that all four retinal layers were present in Ptdsr -knockout mice at E16.5 (Figure 4b,f ). At E18.5 (Figure 4c,g ) and in neonatal animals (postnatal day P0; Figure 4d,h ), the differences in retinal differentiation between Ptdsr +/+ and Ptdsr -/- mice were still evident, but the size reduction of the retinal layers was less pronounced in the knockout mice. Ptdsr -deficient animals seem to have compensated for the marked delay in cellular differentiation and expansion of retinal layers. Close examination of retinal structures revealed that the inner granular layer was still less expanded in Ptdsr -deficient animals, however, and that it contained fewer cells and was still severely underdeveloped in comparison with the corresponding retinal layer in control animals (Figure 4c,4g and 4d,4h ). Thus, even mildly affected Ptdsr -/- mutants had ocular malformations with defects in differentiation of retinal structures. We next examined Ptdsr -/- embryos that displayed unilateral or bilateral absence of eyes (Figure 5a ) by serial sectioning of whole embryos. These embryos showed complex malformations of the optical cup, including absence of the lens (Figure 5b ). Most surprisingly, we found pigmented epithelial cells in the nasal cavity of all Ptdsr -knockout mice with anophthalmia that were analyzed histopathologically. We could identify black-colored pigmented cells embedded in the epithelium of the maxillary sinus that resembled presumptive retinal-pigmented epithelium (Figure 5b,c ). Examination of consecutive serial sections revealed the formation of a primitive eye structure, with induction and subsequent proliferation of ectopic mesenchymal tissue immediately adjacent to the displaced pigmented epithelium (Figure 5d ). This structure was clearly induced ectopically, and we failed to identify similar changes in any of the wild-type embryos. In summary, we observed a wide range of ocular malformations in Ptdsr -deficient mice that ranged from differentiation defects in retinal cell layers (for example, the inner granular layer) in mildly affected homozygotes to anophthalmia in severely affected Ptdsr -/- mice that was associated with induction of ectopic eye structures in nasal cavities. Phagocytosis and clearance of apoptotic cells is normal in Ptdsr -deficient mice We next tested whether Ptdsr is functionally required for the clearance of apoptotic cells. We started with an investigation of cell death in vivo in the interdigital areas of the developing limbs. Apoptosis of interdigital cells in the distal mesenchyme of limb buds occurs most prominently from developmental stages E12.0 to E13.5 and can be easily examined in situ by whole-mount terminal deoxynucleotide transferase-mediated UTP end-labeling (TUNEL). We compared the pattern of interdigital cell death in fore and hind limb buds from Ptdsr -/- ( n = 3) and Ptdsr +/+ ( n = 3) mice at E12.5 and E13.5. No differences in accumulation of TUNEL-positive cell corpses were observed between the two genotypes (Figure 6a ). The kinetics of cell death occurrence and regression of the interdigital web was similar in wild-type and mutant littermates, providing no evidence that Ptdsr -deficiency is associated with impaired clearance of apoptotic interdigital cells during limb development. To investigate further whether removal of apoptotic cells is impaired in Ptdsr -/- mice, we stained immunohistochemically for activated caspase 3 (aCasp3) and analyzed additional organs and tissues where apoptosis plays a crucial role in tissue remodeling during development. Starting at E12.5, we analyzed and compared the number and distribution of aCasp3-positive cells in over 140 serial sections of three wild-type and six Ptdsr -/- embryos in consecutive and corresponding sections. The sagittal sections were separated by 5 μm, allowing a detailed analysis of apoptosis in several organs and tissues. Tissue restructuring by programmed cell death occurred most notably within the ventral part of the neural tube (Figure 6b,f ) and in the developing paravertebral ganglia (Figure 6d,h ) with many apoptotic cells being present. In these tissues Ptdsr is highly expressed at E12.5 (Figure 2c ) but we observed no difference in the number or distribution of apoptotic cells in Ptdsr +/+ and Ptdsr -/- embryos. The same was true for the developing kidney: apoptotic cells were present in Ptdsr +/+ and Ptdsr -/- embryos, in limited numbers, but we failed to detect any differences in the number of apoptotic cells between the genotypes (Figure 6c,6g ). Furthermore, when we continued our analysis of apoptotic cell clearance in vivo at E16.5, E17.5 and E18.5 of embryonic development as well as in neonatal mice, the number and distribution of apoptotic cells was similar in both genotypes. As already observed at E12.5, analysis of aCasp3-stained sections of the developing thymus, heart, diaphragm, genital ridge, eyes and retina convincingly showed that there was no impairment in apoptotic cell removal in Ptdsr -/- mice. Moreover, because Li and colleagues [ 31 ] reported impaired clearance of dead cells during lung development in Ptdsr -deficient mice, we examined the rate of apoptosis induction and cell clearance in our Ptdsr -knockout mice in the lung. Analysis of aCasp3-stained lung tissue from Ptdsr +/+ and Ptdsr -/- mice at E17.5 and P0 demonstrated that apoptosis was an extremely rare event during lung morphogenesis at this stage. In addition, there were no differences in the number or distribution of apoptotic cells in Ptdsr -/- and Ptdsr +/+ mice. Furthermore, we were unable to detect any evidence of tissue necrosis in lungs from Ptdsr -deficient mice. In contrast to the report of Li et al. [ 31 ], we never observed recruitment of neutrophils or other signs of pulmonary inflammation at any stage of development in our Ptdsr -deficient mice. To analyze whether macrophages are recruited into areas where apoptosis is prominent during embryogenesis, we stained consecutive serial sections either with the macrophage surface marker F4/80 or with aCasp3. Surprisingly, there was no co-localization of macrophages with apoptotic cells. In virtually all embryonic tissues, apoptotic cells and macrophages were localized in different compartments (Figure 6e,6i ; and see also Additional data file 1, Figure S1). This suggests that at this stage of development it is mainly neighboring cells that are involved in removal of apoptotic cells, rather than professional macrophages. In summary, our analysis in vivo did not reveal any impairment in apoptotic cell clearance in Ptdsr -deficient embryos during development and further suggests that phagocytosis of apoptotic cells is mainly mediated by non-professional 'bystander' cells. To determine whether macrophages from Ptdsr -knockout mice were impaired in the efficacy of apoptotic cell uptake in vitro , we performed phagocytosis assays with fetal-liver-derived macrophages (FLDMs) and quantified their phagocytosis rates. Phagocytosis of apoptotic thymocytes was investigated at 60, 90 and 120 minutes after addition of target cells in the absence of serum. Analysis of phagocytosis rates by flow cytometric analysis (FACS) revealed no differences in the efficacy of apoptotic cell uptake between Ptdsr -/- and Ptdsr +/+ macrophages and demonstrated no differences in apoptotic cell engulfment between selected time points (data not shown). To re-examine and further independently validate the result of normal apoptotic cell uptake by Ptdsr -/- macrophages, we performed phagocytosis assays for 60 min and determined the percentage of macrophages that had engulfed apoptotic cells, in a total of at least 300 macrophages counted by fluorescence microscopy. Phagocytosed, 5-carboxytetramethylrhodamine- (TAMRA-) labeled apoptotic cells were identified as being engulfed by inclusion in F4/80-labeled macrophages. Analysis was done independently by three investigators who were not aware of macrophage genotypes ( Ptdsr -/- or Ptdsr +/+ ). Again, no differences were found in the percentage of macrophages that had engulfed apoptotic cells (Figure 7a,c,e ) or in the relative number of phagocytosed apoptotic cells per macrophage (phagocytotic index; Figure 7f ). Moreover, single Ptdsr -/- macrophages could be identified that had engulfed even more apoptotic target cells than had wild-type macrophages (Figure 7b,d ). Thus, Ptdsr -deficient macrophages had a normal ability to ingest apoptotic cells and were not impaired in recognition or phagocytosis of cells that had undergone programmed cell death. Ptdsr -deficiency results in reduced production of pro- and anti-inflammatory cytokines after macrophage stimulation In addition to its suggested importance for phagocytosis of apoptotic cells, it has been proposed that Ptdsr fulfils a second crucial role in regulating and maintaining a non-inflammatory environment upon the recognition of apoptotic cells by macrophages [ 26 ]. We therefore tested whether Ptdsr -/- macrophages were able to release anti-inflammatory cytokines after ingestion of apoptotic cells. We examined levels of TGF-β1 and interleukin-10 (IL-10) after stimulation of FLDMs with lipopolysaccharide (LPS), with and without co-culture of apoptotic cells. Quantification of TGF-β1 and IL-10 levels after 22 hours of culture demonstrated that Ptdsr -/- macrophages were able to secrete these anti-inflammatory cytokines upon ingestion of apoptotic cells, although at a slightly lower level than wild-type (Figure 8a,b ). This indicates that ablation of Ptdsr function does not compromise in general the ability of macrophages to release immune-suppressive cytokines after recognition and engulfment of apoptotic cells. To analyze whether pro-inflammatory signaling is affected in Ptdsr -/- macrophages, we stimulated FLDMs from Ptdsr +/+ and Ptdsr -/- mice with LPS and measured levels of tumor necrosis factor-α (TNF-α) at different time points after stimulation (Figure 8c ). Ptdsr -/- macrophages produced significantly less TNF-α than did wild-type macrophages. The difference in TNF-α secretion was first visible after 3 h of LPS stimulation and became more prominent during the course of the experiment (for example, after 9 h and 12 h of LPS stimulation; Figure 8c ). To analyze whether TNF-α release by Ptdsr -/- macrophages can be affected by engulfment of apoptotic cells, we stimulated FLDMs with LPS, apoptotic cells or both. Quantification of TNF-α levels by ELISA after 22 h showed that Ptdsr -deficient macrophages release less TNF-α after stimulation with LPS alone, and also after double stimulation of macrophages with LPS and apoptotic cells (Figure 8d ). Moreover, the double stimulation demonstrated that the LPS-induced TNF-α release by Ptdsr -/- macrophages could be inhibited by co-administration of apoptotic cells to an extent comparable to that seen in wild-type macrophages. Similar results were obtained when other pro-inflammatory cytokines, such as interleukin-6 and monocyte chemoattractant protein-1, were analyzed (data not shown). These results indicate that Ptdsr is not required in macrophages for the inhibition of pro-inflammatory signaling after recognition and engulfment of apoptotic cells. Ptdsr -deficiency does, however, affect the overall release of pro- and anti-inflammatory cytokines after stimulation with LPS and after double treatment with LPS and apoptotic cells, indicating that Ptdsr -deficient macrophages have a reduced capacity to produce or secrete pro- and anti-inflammatory cytokines. Discussion Ptdsr is required for the differentiation of multiple organ systems during development In this study, we have generated a null mutation in the phosphatidylserine receptor ( Ptdsr ) gene in C57BL/6J mice. We show that ablation of Ptdsr results in profound differentiation defects in multiple organs and tissues during embryogenesis, although with variable penetrance. While this work was in progress, two other groups reported the generation of Ptdsr -deficient mice [ 31 , 32 ]. In all three knockout mouse lines, the first two exons ([ 31 ] and this study) or exons one to three [ 32 ] were deleted by replacement with a neomycin-selection cassette. The Ptdsr -knockout mouse lines differ in the genetic background in which the mutation was generated and maintained, however. In our case, the Ptdsr -null allele was generated in an isogenic C57BL/6J background, whereas Li et al. [ 31 ] and Kunisaki et al. [ 32 ] investigated the phenotype of their Ptdsr -knockout mice in a mixed 129 × C57BL/6 background. The ablation of Ptdsr function results in perinatal lethality in all cases, but there are interesting differences in severity or expressivities of phenotypes among the different Ptdsr -deficient mouse lines. This might be due either to differences in genetic background or because the phenotypes that have been investigated in this study have not been analyzed in such detail before. In the Ptdsr -knockout mouse line reported here, growth retardation started from E12.5 onwards and was associated with delayed differentiation in several organs in which Ptdsr is expressed either during embryogenesis or later in adulthood. At E16.5 almost no branching morphogenesis of the lung epithelium was observed in Ptdsr -/- lungs. Similarly, epithelial structures were only partially developed in mutant kidneys, without terminal differentiation of Bowman's capsule and with a severe reduction in the number of differentiated collecting tubules. Likewise, the differentiation of the intestine was also severely delayed at this developmental stage. When compared with wild-type controls, intestinal tissues of Ptdsr knockout mice appeared unstructured, with an absence of enteric ganglia and of differentiated smooth muscle tissue. Interestingly, defects in kidney and intestine differentiation were not described in the Ptdsr -knockouts generated by Li et al. [ 31 ] and Kunisaki et al. [ 32 ]. Surprisingly, when we examined Ptdsr -/- embryos shortly before birth (E18.5) or neonatally, we found only mild differentiation delays in organs that appeared severely affected at mid-gestation. This 'recovery' was most visible in Ptdsr -/- lungs: at P0 we found expanded lungs in the knockout mice that showed normal branching patterns, with differentiated alveoli and bronchioles. We investigated the occurrence of programmed cell death during lung development in wild-type and Ptdsr -knockout mice throughout embryogenesis (E16.5 to P0). Comparative immunohistochemistry for aCasp3 revealed that apoptosis is a rare event during lung morphogenesis. Furthermore, we failed to detect any differences in the number of apoptotic cells in Ptdsr -knockout and wild-type animals in the rare cases where we could detect apoptotic cells within lung tissues. These findings are contrary to the results reported by Li et al. [ 31 ], who suggested that impaired clearance of apoptotic mesenchymal and epithelial cells causes a failure in lung morphogenesis in Ptdsr -deficient mice. In contrast, our findings are in line with the current view on lung development during embryogenesis. Accordingly, formation of the epithelial lung via branching morphogenesis can be subdivided into a series of sequential steps that involve: first, formation of the organ anlage in the form of a placode; second, primary bud formation by placode invagination; third, branch initiation and branch outgrowth; fourth, further reiteration of the branching process; and fifth, terminal differentiation of organ-specific proximal and distal structures [ 34 , 35 ]. In contrast to other invagination processes during embryogenesis, such as mammary gland formation, the lumen of the lungs is expanded by successive branching events, branch outgrowth and elongation, rather than by apoptosis [ 34 , 36 ]. Finally, because the lungs of Ptdsr -/- neonates were almost fully expanded and appeared normal in structure in comparison to wild-type littermates, it is highly unlikely that Ptdsr mutants die of respiratory lung failure. In addition, Li and colleagues [ 31 ] demonstrated that surfactant expression is normal in Ptdsr -deficient animals, supporting the idea of normal maturation of surfactant-producing type II alveolar epithelial cells and lung function. Other defects must therefore be responsible for the death of Ptdsr -mutant mice. The frequently observed subcutaneous edema of various extents in Ptdsr -deficient homozygotes gave us a hint that Ptdsr -deficiency and lethality might be associated with cardiovascular problems. Indeed, very recently we have obtained strong evidence that Ptdsr -knockout mice die as a result of defects in heart development that are associated with specific cardiopulmonary malformations; (J.E. Schneider, J.B., S.D. Bamfort, A.D.G., C. Broadbent, K. Clarke, S. Neubauer, A.L. and S. Battacharya, unpublished observations). In addition, we demonstrate that eye development requires a functional Ptdsr gene. Ptdsr -deficient embryos can be roughly divided into two categories. The first, severely affected group develops anophthalmia that correlates with formation of ectopic retinal-pigmented epithelium and induction of proliferation of underlying mesenchyme in the nasal cavity. This phenotype represents a completely novel lesion that to our knowledge has not been described before in any other mouse mutant. The second group shows normal external eye structures, although in this case retinal development is temporally delayed during mid-gestation, with persistent, abnormal morphogenesis of the inner granular retinal layer at later stages of embryogenesis. A possible explanation for these two phenotypes can be found in the expression pattern of the Ptdsr gene. Initially, Ptdsr is expressed throughout the whole developing nervous system, with exceptionally high levels in the anterior part of the forebrain. Later expression becomes more restricted to the developing retina and lens. Thus, Ptdsr might play an important role in early events of ocular morphogenesis, such as establishment and bisection of eye fields and formation of optic cups. These early eye-formation steps are closely interconnected with development of the forebrain [ 37 , 38 ] and the nose [ 39 - 41 ]. Interestingly, we occasionally observed serious malformations of forebrain and nasal structures in Ptdsr -knockout embryos that were associated with bilateral anophthalmia (see for example the mutant embryo in Figure 1g ). This suggests that Ptdsr is involved in the regulation of differentiation processes within forebrain regions, and that ablation of Ptdsr function might secondarily affect early eye formation. Li et al. [ 31 ] found smaller lenses in Ptdsr -knockout mice and described the formation of retinal protrusions, although anophthalmia and specific differentiation defects of retinal cell layers were not reported in their study. Li et al. proposed [ 31 ] that the eye phenotype they observed could be explained by failed removal of apoptotic cells during eye development, but we think that the observed defects are unrelated to a failure of apoptotic cell clearance. A recent comprehensive kinetic analysis of apoptosis induction during mouse retinal development described four major peaks of apoptotic cell death [ 42 ]. This study demonstrated that there is an initial phase of cell death during the invagination of the optic cup (E10.5), followed by subsequent waves of apoptosis induction immediately before and after birth (E18.5 to postnatal day P2), and from postnatal days P9 to P10 and P14 to P16 [ 42 ]. Thus, besides the formation of the inner and outer layers of the optic cup in early eye development, other major phases of retinal cell apoptosis take place only postnatally and correspond to important periods in the establishment of neuronal connections. Furthermore, cell death during normal retinal development occurs in retinal layers distinct from the inner granular layer where we observed the most pronounced differentiation defects in the Ptdsr -/- mutants described here. Other studies that connect the postnatal elimination of apoptotic photoreceptor cells to Ptdsr-mediated macrophage engulfment [ 43 ] should be interpreted with extreme caution as these studies were based on the monoclonal anti-Ptdsr antibody mAb 217G8E9 [ 26 , 43 ] (see below). Consistent with the results of Li et al. [ 31 ], we found particular brain malformations in our Ptdsr -/- mice. Exencephaly and hyperplastic brain phenotypes were observed at a low penetrance in Ptdsr- mutant mice (less then 4.5% of homozygotes), but these do not resemble to any extent the brain-overgrowth phenotypes of caspase- or Apaf1 -knockout mice ([ 44 ], and references therein) in that we failed to identify any differences in the number or distribution of apoptotic cells or pyknotic cell clusters in the neuroepithelium of Ptdsr -/- and Ptdsr +/+ mice. Thus, reduced cell death or diminished clearance of apoptotic neural progenitor cells is unlikely to be the cause of the brain hyperplasia. In summary, our studies demonstrate that Ptdsr is required for normal tissue differentiation, especially during the mid-gestation period when we observed the most severe differentiation delays in several organs of Ptdsr -knockout mice. The multiple defects in tissue differentiation cannot be explained by failure of apoptotic cell clearance, as this process is normal in our Ptdsr -knockout line. This result therefore indicates that Ptdsr has a novel, hitherto unexpected, role in promoting tissue maturation and terminal differentiation. Additional studies with conditionally targeted Ptdsr -deficient mice are required to investigate the role of spatial and temporal Ptdsr expression and function during tissue differentiation. Ptdsr is not essential for the clearance of apoptotic cells Our studies demonstrate that Ptdsr is not a primary receptor for the uptake of apoptotic cells. Investigation of apoptotic cell clearance in vivo in Ptdsr -/- embryos conclusively showed that removal of apoptotic cells is not compromised by ablation of Ptdsr function. Comparative analysis of ten different tissues and organs in Ptdsr +/+ and Ptdsr -/- animals at several stages of embryonic development and in neonates failed to identify impaired uptake of apoptotic cells at any time during development. Furthermore, phagocytosis assays in vitro demonstrated a completely normal uptake of apoptotic cells by Ptdsr -/- macrophages, with some knockout macrophages showing loads even higher than wild-type of engulfed dead cells. These results are contrary to the expected role of Ptdsr in apoptotic cell clearance and to the reported findings of Li et al. [ 31 ] and Kunisaki et al. [ 32 ], as well as to a study done with a phosphatidylserine receptor null allele in C. elegans [ 45 ]. In previous studies in the mouse, the distribution and amount of apoptotic cells in Ptdsr -knockout and control animals were investigated in only a few tissues and at one [ 31 ] or two [ 32 ] developmental stages. Li et al. [ 31 ] examined lung, midbrain and retina at day E17.5 of gestation and identified apoptotic cells by TUNEL staining. Their findings must be interpreted with caution because remodeling of cellular structures by apoptosis in specific retina layers is known to occur mainly postnatally [ 42 ], and apoptosis plays an important physiological role in the maintenance and homeostasis of lung epithelium after birth or in pathological conditions involving pulmonary inflammation and not during lung development [ 46 ]. This postnatal role for apoptosis is in accordance with our data, as we rarely observed apoptotic cells in retina or lung tissue throughout embryogenesis in Ptdsr +/+ and Ptdsr -/- mice. Kunisaki et al. [ 32 ] analyzed TUNEL-stained sections of liver and thymus at days E13.5 and E16.5 of development in Ptdsr +/- and Ptdsr -/- embryos and found reduced rather than increased numbers of TUNEL-positive cells in Ptdsr -deficient embryos. Using co-localization of TUNEL-positive cells with F4/80-positive macrophages they suggested that Ptdsr -/- embryos exhibited a three-fold increase in the frequency of unphagocytosed TUNEL-positive cells together with a severely reduced number of F4/80-positive cells. These results must be interpreted very carefully, however, as it is technically difficult to unambiguously identify engulfed target cells in individual macrophages in solid tissues by fluorescence microscopy. In addition, our data suggest that during embryogenesis, macrophage-mediated clearance of apoptotic cells is not the only - or even the primary - mechanism for the removal of apoptotic cells. In many tissues where programmed cell death occurs as a prominent event during embryogenesis, such as remodeling of the genital ridge during gonad morphogenesis and differentiation of the neural tube, we found almost no co-localization of apoptotic cells and macrophages. This indicates that in these cases clearance of apoptotic cells is directly mediated by neighboring 'bystander' cells rather than by macrophages that have been recruited into areas where apoptosis occurs. Obviously these in vivo clearance mechanisms are not compromised by Ptdsr -deficiency in our knockout mutant. This finding is in line with studies in macrophageless Sfpi1 -knockout embryos that are deficient for the hematopoietic-lineage-specific transcription factor PU.1. Here, the phagocytosis of apoptotic cells during embryogenesis is taken over by 'stand-in' mesenchymal neighbors [ 47 ]. As recognition of phosphatidylserine is thought to be a universal engulfment mechanism for all cells that are able to phagocytose apoptotic cells, it is very striking that apoptotic cell clearance mediated by non-professional bystander cells is also not compromised by Ptdsr -deficiency. In contrast to Li et al. [ 31 ], we did not observe any impairment in the uptake of apoptotic cells by Ptdsr -/- macrophages in vitro . We performed phagocytosis assays in vitro with fetal-liver-derived macrophages, while in their assays, Li and colleagues used thioglycollate-elicited peritoneal macrophages after adoptive transfer of Ptdsr -/- hematopoietic stem cells. The different results obtained in the two studies are puzzling; they might be due to the use of different macrophage or cell populations. We and Kunisaki et al. [ 32 ] found that Ptdsr -deficiency is to some extent associated with defects in hematopoiesis. Thus, it seems possible that recruitment and activation/differentiation of macrophages after adoptive transfer and thioglycollate elicitation are affected by Ptdsr -deficiency. We do not think that the different results observed in Ptdsr -knockout mice in a mixed C57BL/6 × 129 background and in a pure C57BL/6J background can be attributed to genetic background effects: comparison of apoptotic cell engulfment efficacies of thioglycollate-elicited macrophages from 129P2/OlaHsd and C57BL/6J mice did not show any differences in apoptotic cell uptake (J.B. and A.L., unpublished observations). Moreover, in contrast to our studies, neither Li et al. [ 31 ] nor Kunisaki et al. [ 32 ] determined phagocytotic engulfment indexes for Ptdsr -deficient macrophages. Interestingly, we observed differences between Ptdsr +/+ and Ptdsr -/- macrophages in the secretion of pro- and anti-inflammatory cytokines after stimulation with LPS and apoptotic cells. This provides evidence that cellular activation and effector mechanisms are impaired in Ptdsr -deleted macrophages. It remains to be determined which classical pathways of macrophage activation and function involve Ptdsr . This is especially important in light of recent findings that demonstrated nuclear localization of the Ptdsr protein [ 29 ]. Most strikingly, the recently published data regarding the genetic ablation or perturbation of phosphatidylserine receptor function in C. elegans are also contradictory. Wang et al. [ 45 ] reported that psr-1 , the C. elegans homolog of Ptdsr , is important for cell-corpse engulfment, whereas psr-1 RNAi studies performed by Arur et al. [ 25 ] yielded, in this respect, no phenotype. Moreover, Wang and colleagues hypothesized on the basis of their data that psr-1 might act to transduce an engulfment signal upstream of Ced-2 (Crk II), Ced-5 (Dock 180), Ced-10 (Rac 1) and Ced-12 (Elmo) in one of the two cell-corpse engulfment pathways in the worm [ 45 ]. But the loss-of-function phenotype of psr-1 mutants and the complementation phenotypes in overexpressing transgenic worms shown by Wang et al. [ 45 ] are rather weak as compared to the classical C. elegans engulfment mutants [ 8 ]. Many previous functional studies that reported a requirement for Ptdsr for the phagocytosis of apoptotic cells used the monoclonal anti-Ptdsr antibody mAb 217G8E9 [ 26 ]. This antibody was used in Ptdsr binding and blocking experiments, as well as in subcellular localization studies, which led to the conclusion that Ptdsr is a transmembrane receptor critical for signal transduction at the engulfment interface. More recently it was used in binding assays to show that the human and worm Ptdsr molecules can recognize phosphatidylserine [ 45 ]. In the course of the study presented here, we stained immunohistochemically for Ptdsr with mAb 217G8E9 on wild-type and Ptdsr -deficient macrophages and fibroblasts (see Additional data file 1, Figure S2 and data not shown). To our surprise, we observed similar staining patterns with cells of both genotypes. Furthermore, using a Ptdsr-peptide array we found that mAb 217G8E9 can bind weakly to a Ptdsr peptide, explaining the original isolation of Ptdsr cDNA clones by phage display [ 26 ]; but the antibody mainly recognizes additional, as-yet unknown, membrane-associated protein(s) (see Additional data file 1, Figure S2). Experiments that have used this antibody should therefore be interpreted with great caution as they might come to be viewed in a different light. Conclusion Our results demonstrate that Ptdsr is essential for the differentiation and maturation of multiple tissues during embryogenesis. Ablation of Ptdsr function results in neonatal lethality and severe defects in the morphogenesis of several organs. The developmental malformations cannot be explained by impaired clearance of apoptotic cells, a process that proved to be normal in Ptdsr -deficient mice. This opens up the possibility either that there is an as-yet unknown Ptdsr receptor, which might act as a primary phosphatidylserine recognition receptor, or that recognition of phosphatidylserine and subsequent apoptotic cell engulfment and anti-inflammatory signaling are mainly mediated through phosphatidylserine bridging proteins and their cognate receptors. Although Ptdsr -/- macrophages were not impaired in their ability to phagocytose apoptotic cells, they showed reduced cytokine responses after stimulation. Further work will be required to determine the molecular mechanisms of these newly recognized Ptdsr functions during development. Materials and methods Construction of the targeting vector and generation of Ptdsr -knockout and gene-trap mice Targeting vector A Ptdsr -containing bacterial artificial chromosome (BAC) clone (GenBank accession number AC091694; RP-23-316F3) was isolated by sequence homology from a C57BL/6J genomic BAC library (RP-23; BACPAC Resources, Oakland, USA). A 14.5 kb Kpn I/ Bam HI fragment containing the entire Ptdsr locus and 5' and 3' flanking regions was subcloned from this BAC clone and a 1.9 kb Rsr II/ Aat II fragment containing exons I and II of the Ptdsr gene was replaced by a 1.2 kb lox P-flanked neomycin-resistance gene cassette ( neo ). Homologous recombination in ES cells and generation of germ-line chimeras Bruce4 ES cells were transfected with Kpn I-linearized targeting vector and selected with G418. ES-cell clones resistant to G418 were isolated and analyzed by Southern blot analysis for homologous recombination events within the Ptdsr locus. Chimeric mice were produced by microinjection of two independent homologous recombinant ( Ptdsr +/- ) ES cells into BALB/c blastocysts and transfer to pseudopregnant foster mothers followed by development to term. Chimeric males were mated with C57BL/6J females. From the two selected ES-cell clones, one successfully contributed to the germ-line. Germ-line transmission of the mutant allele was verified by PCR and Southern blot of genomic DNA from black coat-color F1 offspring. Ptdsr gene-trap and generation of germ-line chimeras An ES-cell line carrying a β-geo gene-trap vector in the Ptdsr locus was identified by searching the BayGenomics database (BayGenomics, San Francisco, USA; [ 48 ]) with the full-length Ptdsr cDNA. A single ES-cell line was identified carrying the gene-trap in intron V, between exons V and VI of the Ptdsr gene. Chimeric mice were generated by microinjection into CB20 blastocysts and transfer to pseudopregnant foster mothers. Chimeric males were mated with 129P2/OlaHsd females. Germ-line transmission of the mutant gene-trap allele was verified by expression analysis using β -galactosidase staining and RT-PCR. Genotype analysis The genotypes of embryos or animals were determined by PCR analysis and confirmed by Southern blot. Genomic DNA for PCR was prepared from extraembryonic membranes or tail clips using a non-organic tail-DNA extraction protocol [ 49 ]. High molecular weight genomic DNA for Southern blotting was prepared according to standard protocols. For PCR analysis the wild-type Ptdsr allele was detected using forward primer 1 (5'-GACACTGTCCATGGCAAACAC-3') and reverse primer 2 (5'-TAAAGTCGCCTTCCAGAAGATT-3'). The primer 1 site is located 5' to the deletion and the primer 2 site within the deletion. This primer pair amplified a fragment of approximately 300 bp from wild-type and Ptdsr +/- mice but not from Ptdsr -/- mutants. To detect the mutant Ptdsr allele, genomic DNA was also amplified using primer 1 and reverse primer 3 (5'-CCACACGCGTCACCTTAATA-3'), which corresponds to a sequence in the neo cassette. In this case, a 500 bp fragment was detected in mice heterozygous or homozygous for the mutant allele, while no signal was detected in wild-type mice. For Southern blot analysis, genomic DNA (30 μg) was digested overnight with Bam HI (30 U; Roche Diagnostics GmbH, Mannheim, Germany) and Sca I (30 U; Roche), fractionated on a 0.8 % agarose gel, transferred to a nylon membrane (Hybond N; Amersham Biosciences Europe GmbH, Freiburg, Germany) and hybridized with 5' and 3' flanking probes. The Bam HI digest was hybridized with a Ptdsr -specific 5' flanking probe, and Southern blot gave a single 17.2 kb band for wild-type ( +/+ ), an 11.6 kb band for homozygous ( -/- ) and both bands for heterozygous ( +/- ) mice. The Sca I digest was hybridized using a 3' flanking probe, and Southern blot gave a single 12.4 kb band for wild-type, a 17.2 kb band for homozygous and both bands for heterozygous mice. Northern blot analysis Total RNA was isolated from homogenized embryos using TRIZOL reagent (Invitrogen GmbH, Karlsruhe, Germany). For northern blots, either total RNA (30 μg) was extracted from embryos, electrophoresed and transferred to a nylon membrane (Hybond N; Amersham) or a polyA + RNA northern blot (OriGene Technologies Inc., Rockville, USA) was hybridized using as the probe a Ptdsr fragment amplified from wild-type cDNA using forward primer 5'-GTTCCAGCTCGTCAGACTCG-3' and reverse primer 5'-TGCCCCTAAGACATGACCAC-3'. In all experiments the same membrane was re-hybridized with a β-actin probe (OriGene) to confirm that equivalent RNA levels were present in each lane. Northern blotting indicated that homozygous mutant embryos did not express Ptdsr mRNA and heterozygous mutant embryos expressed only reduced amounts of Ptdsr mRNA. Western blot analysis Embryos (E13.5) for protein isolation were homogenized in lysis buffer containing 1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (CompleteMini; Roche). Equal amounts (25 μg) of protein lysate were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore, Billerica, USA) according to standard protocols. Western blots were done using a specific antibody to Ptdsr (PSR N-20, sc-11632; Santa Cruz Biotechnology Inc., Santa Cruz, USA) and β-actin (ab-6276; Abcam, Cambridge, UK) as described by the supplier. Secondary antibodies conjugated to horseradish peroxidase were from Santa Cruz and Abcam, used as described by the supplier, and detection was performed with an enhanced chemiluminescence system (ECLPlus; Amersham). Animal experiments Wild-type C57BL/6J and 129P2/OlaHsd mice were obtained from Jackson Laboratories (Bar Harbor, USA) and Harlan UK (Bicester, UK), respectively. All mice were housed in individually ventilated cages in a specific pathogen-free environment with a 12 h light-dark cycle and were fed a regular unrestricted diet. The GBF's routine surveillance program screened for selected pathogens. The Ptdsr tm1Gbf mutant was crossed to C57BL/6J mice to establish the co-isogenic C57BL/6J- Ptdsr tm1Gbf mouse line. All studies were approved by the appropriate authorities. Isolation of embryos Heterozygous male and female mice were intercrossed in order to obtain Ptdsr -deficient progeny. Females were daily monitored for vaginal plugs, and noon of the day of plug detection was defined as E0.5. Embryos at indicated time points were dissected in sterile PBS, washed in ice-cold PBS and transferred to cold fixative. Extra-embryonic membranes were kept and used for genotyping. Ptdsr -/- embryos and their wild-type littermates were used for experiments. Histology, TUNEL staining and immunohistochemistry Embryos for histology and immunohistochemistry were harvested and fixed in 10% neutral-buffered formalin, dehydrated through a graded series of alcohol, embedded in paraffin, sagittally sectioned at 5 μm intervals, and every fifth section was processed for hematoxylin and eosin (H&E) staining according to standard protocols. Remaining sections of wild-type and Ptdsr -/- specimens were used for immunohistochemistry. For detection of apoptotic cells and macrophages, anti-aCasp3 (an antibody specific for activated caspase 3; R&D Systems, Minneapolis, USA) and anti-F4/80 (Serotec GmBH, Düsseldorf, Germany; #MCA 1957) antibodies were used as described by the supplier. Detection was performed using indirect streptavidin with biotinylated secondary antibodies and cobalt-enhanced diaminobenzidine (brown) or fast-red (red) as chromogens. Sections were counterstained with hematoxylin. For whole-mount terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL), limb buds were dissected from E12.5 and E13.5 embryos, fixed in 4% paraformaldehyde and processed for analysis as previously described [ 50 ]. Preparation of fetal liver-derived macrophages (FLDMs) Fetal livers were excised from embryos at E12.5 and E13.5, respectively, washed in PBS and dissociated enzymatically for 60 min at 37°C. The digestion buffer (150 μl per liver) comprised 0.6 U/ml dispase I (Roche), 0.1% collagenase D (Roche), 10 U DNase (Roche), and 20% FCS in PBS. X-Vivo 15 medium (Cambrex, East Rutherford, USA) was added to the resulting cell suspension, and after centrifugation (200 × g ; 3 min) cells were resuspended in X-Vivo 15 medium supplemented with 50 ng/ml macrophage colony-stimulating factor (M-CSF; Sigma-Aldrich, St. Louis, USA) and cultured on non-treated tissue-culture dishes at 37°C with 5% CO 2 . Every second or third day the medium was changed by centrifugation. Following withdrawal of M-CSF on day 6 after excision, adherent cells were cultured for an additional 24-48 h in X-Vivo 15 medium. Macrophage phagocytosis assays For preparation of monolayer cultures of macrophages, FLDMs were plated on glass coverslips in 24 well plates (2 × 10 5 cells per well) in X-Vivo 15 medium. For preparation of apoptotic target cells, primary thymocytes were harvested from the thymus of 4- to 8-week-old C57BL/6J mice, stained with TAMRA for 15 min, and apoptosis was induced either by treating cells with 5 μM staurosporine in medium for 4 h at 37°C or by culturing cells in medium overnight. The efficacy of apoptosis induction was compared in thymic target cells and controls by FACS analysis. On average, 60% of the cells of the resulting population were apoptotic, with exposed PS on their surface, and less than 5% of the cells were necrotic, as confirmed by FITC-annexin V and propidium iodide staining. The apoptotic thymocytes obtained were washed with PBS and added to the prepared FLDM cultures (ratio 10:1). Phagocytosis was then allowed to proceed at 37°C and 5% CO 2 . After the indicated time periods, the uptake of apoptotic cells by FLDMs was stopped by intensive washing of co-cultures with cold PBS to remove unphagocytosed cells. To measure phagocytosis of apoptotic thymocytes, macrophages were further processed for immunofluorescence analysis. Cells were fixed in 4% paraformaldehyde, blocked in 0.5% BSA/PBS and stained with an anti-F4/80 antibody (Serotec) followed by a secondary antibody coupled to Alexa 488 (Molecular Probes Inc., Eugene, USA). Coverslips were mounted on slides and engulfed thymocytes were enumerated by fluorescence microscopy. The percentage of phagocytosis was calculated by counting at least 300 macrophages and determining the number of macrophages that had engulfed apoptotic thymocytes. The phagocytotic index was calculated according to the following formula: phagocytotic index = (total number of engulfed cells/total number of counted macrophages) × (number of macrophages containing engulfed cells/total number of counted macrophages) × 100. The experiments were performed at least three times, each time in triplicate, and the counting was done by three different investigators. Measurement of macrophage cytokine production Monolayer cultures of FLDMs and apoptotic thymocytes were prepared as described above. FLDMs were incubated with medium, LPS (10 ng/ml), apoptotic cells (ratio 1:10) or both for the determination of IL-10, TGF-β1 or TNF-α levels after co-culture for 22 h. For TNF-α quantification at various time points, FLDMs were cultured with a high concentration of LPS (100 ng/ml). Culture supernatants were harvested and TNF-α (Mouse TNF-α OptEIA set; BD Biosciences, Heidelberg, Germany) and TGF-β1 (Quantikine, TGF-β1 immunoassay; R&D Systems) were measured by ELISA as described by the supplier. IL-10 in culture supernatants was determined by a cytometric bead assay (Mouse inflammation CBA; BD Biosciences) as indicated in the manual. Data are presented as mean ± SEM from at least three independent experiments, each carried out in triplicate. Analysis of the results used the Wilcoxon-signed rank test; p values below 0.05 were considered significant. Additional data files Additional data file 1 contains: Figure S1 showing the localization of apoptotic cells and macrophages in the subcutis of developing embryos; and Figure S2 showing immunohistochemical staining of the Ptdsr protein in macrophages derived from wild-type and Ptdsr -knockout mice. Supplementary Material Additional data file 1 Figure S1 showing the localization of apoptotic cells and macrophages in the subcutis of developing embryos; and Figure S2 showing immunohistochemical staining of the Ptdsr protein in macrophages derived from wild-type and Ptdsr -knockout mice Click here for additional data file | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549712.xml |
497053 | What is the role of theory in health behavior change interventions? | null | The discussion about the interplay between theory and intervention has a long history and much has been written and published. The companion perspective papers by Rothman and Jeffery provide alternative points of view on this important and complex question. Their papers are not intended to be critical reviews of the literature. Instead, the aims of the two papers are to delineate the problem, present the different perspectives of the authors on the issue, and suggest constructive steps researchers can take to move the field forward. We hope that these articles generate a lively response from our readers and spur innovative ideas for strategies to cultivate a healthy developmental interplay between theory and intervention in behavioral nutrition and physical activity research. We invite you to consider the intellectual and practical challenges posed by the two authors and perhaps respond with your perspectives, which may be published in the IJBNPA. We look forward to hearing from you soon. Simone A. French, Ph.D. Tony Worsley, Ph.D. Co-Editors, IJBNPA | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC497053.xml |
521492 | Seal of transparency heritage in the CISMeF quality-controlled health gateway | Background It is an absolute necessity to continually assess the quality of health information on the Internet. Quality-controlled subject gateways are Internet services which apply a selected set of targeted measures to support systematic resource discovery. Methods The CISMeF health gateway became a contributor to the MedCIRCLE project to evaluate 270 health information providers. The transparency heritage consists of using the evaluation performed on providers that are referenced in the CISMeF catalogue for evaluating the documents they publish, thus passing on the transparency label from the publishers to their documents. Results Each site rated in CISMeF has a record in the CISMeF database that generates an RDF into HTML file. The search tool Doc'CISMeF displays information originating from every publisher evaluated with a specific MedCIRCLE button, which is linked to the MedCIRCLE central repository. Starting with 270 websites, this trust heritage has led to 6,480 evaluated resources in CISMeF (49.8% of the 13,012 resources included in CISMeF). Conclusion With the MedCIRCLE project and transparency heritage, CISMeF became an explicit third party. | Background The availability of Internet health tools and services has been increasing at a phenomenal rate in recent years making the Internet a major source of knowledge for healthcare professionals, medical students and also patients and the general public. This increase has made it an absolute necessity to continually assess the quality of health information on the Internet. Indeed, creating a Web site is relatively easy, therefore uncontrolled health information can be launched by virtually anyone with access to the Internet. Peer review is often absent throughout this media as opposed to scientific journals. There have been numerous debates about the variable quality of health information on the Internet and its impact on public health [ 1 ]. There is no other field in which inaccurate, incomplete, or biased information can be potentially more damaging [ 2 ]. In the past five years, a lot of authors have scrutinized the quality of the health content available on the Internet. These studies, assessing the quality of health information, have been extensively reviewed by Eysenbach et al. [ 1 ]. In the meantime, several worldwide initiatives have been undertaken to define criteria for assessing the quality of health information on the Internet. These initiatives have been reviewed by Risk and Dzenowagis [ 3 ]. However, no consensus has been reached by healthcare professionals or consumers on how to assess the quality of health information on the Internet. As of today, access to accurate and trustworthy health information on the Internet is not an easy task; there are a great number of directories and search engines available in this new media [ 4 ]. But, there is also a need to develop reliable and quality-controlled health subject gateways to disseminate relevant trustworthy health information. Koch [ 5 ] defined quality-controlled subject gateways as Internet services which apply a rich set of quality measures to support systematic resource discovery. Considerable manual effort is used to process a selection of resources, which meet quality criteria, and to display a rich description and indexing of these resources with standards-based metadata. Regular checking and updating ensure optimal collection management. The main goal is to provide a high-quality of subject access through resource indexing using controlled vocabularies and via a practical classification structure for advanced searching and browsing. The objective of CISMeF (French acronym for Catalogue and Index of health resources in French) [ 6 , 7 ] is to describe and index the main health resources in French in order to assist health professionals, students and consumers in their search for electronic information available on the Internet. CISMeF is a quality-controlled subject gateway initiated by the Rouen University Hospital (RUH). Each of the following phases proposed by Koch [ 5 ], which characterise a typical quality-controlled subject gateway, are implemented in CISMeF: (a) selection and collection development, based on the Net Scoring, a list of 49 criteria to assess quality of health information (URL: ) [ 4 ], (b) collection management, (c) creation of metadata (performed by experts), (d) resource description (an extensive and documented metadata set), and (e) resource indexing (using a controlled vocabulary system). CISMeF is manually maintained. In CISMeF, a resource is defined as 1) a Web site or 2) high-quality documents from this Web site. CISMeF describes and indexes the most important sources of institutional health information in French, in order to allow them to be searched quickly and precisely. A great variety of resources are indexed, in terms of resource type (clinical guidelines, teaching material, patients information, etc.), and resource format (html, pdf, etc.). Its Universal Resource Locator (URL) is The CISMeF Web site opened in February 1995. In December 2003, 13,012 resources had been indexed, with an average of 55 new resources indexed each week. CISMeF is considered by most professionals as the reference health institutional Web site in France with as many as 25,000 unique machines visiting this Web site by working day. Doc'CISMeF is the search tool of the CISMeF gateway [ 7 ]. In 1997, because the quality of health information became an important issue for the building and maintenance of a trustworthy health gateway, the CISMeF team participated in the development of a user guide named Net Scoring [ 8 ]. The goal of the Net Scoring project was to provide a set of criteria that can be consistently used to assess the quality of health information on the Internet. Between 1997 – 2002 the CISMeF gateway selected health resources using the main criteria established by the Net Scoring initiative (source of information, disclosure, editorial review process, date of last update, and feedback mechanism) in view of the fact that the selection process is mandatory to create a trustworthy health Internet directory. Resources that are not compliant with basic ethical criteria are not included in the CISMeF database. In 2002, CISMeF became a contributor to the MedCIRCLE project (URL: ) [ 9 ]. The aim of this project is to establish a global Web of transparency for networked health information and to increase the accessibility and findability of trustworthy health websites using "Semantic Web" approaches, which essentially means to make "narrative" information on the Web accessible in a machine processable format by using RDF (Resource Description Framework) expressed in XML (eXtended Markup Language) [ 10 ]. MedCIRCLE is a collaboration of trustworthy European health subject gateways, medical associations, accreditation, certification, or rating services, which share the common goal of evaluating, describing, or indexing health information. MedCIRCLE began in March 2002 and lasted till December 2003. Whereas CISMeF initially addressed quality on a more finely grained level, i.e., the quality of documents or single web pages, MedCIRCLE focused on whole websites including information about the respective publisher. The main deliverable of this project for the CISMeF team was the evaluation and rating of 270 health information providers (or publishers) which who release health resources in French on a regular basis. Because CISMeF catalogues and indexes not only Web sites but also and mainly quality-controlled documents from those health publishers, we introduced the concept of "transparency transitivity" or " transparency heritage". It consists in applying to these documents the evaluation performed for their publishers, thus passing on the transparency label from the publishers to their documents. Methods Providing transparency related metadata Health professionals have begun to realize that it is their responsibility to guide consumers and patients to the best available medical information on the web. Many national governments and medical societies have acknowledged that it is their responsibility to help users to identify "good quality" information sources and have begun to develop national health gateways (such as HealthinSite in Australia, NHS Direct in the UK, or Healthfinder in the USA), portal sites and other forms of "infomediaries" such as seals of approval [ 2 ] or certification mechanisms in an effort to help consumers to locate trustworthy information resources. However, current approaches do not harness any of the advantages of the Web as a decentralized, distributed information system. There is a need for "next generation" tools, including intelligent knowledge-based tools, allowing consumers to positively and actively identify reliable health information that suits their needs. The three application partners of MedCIRCLE, besides CISMeF in France, were the Agency for Quality in Medicine (AQuMed) in Germany and the Official Medical College of Barcelona (COMB). AQuMed was founded in March 1995 as a joint institution of the German Medical Association and the National Association of Statutory Health Insurance Physicians. AQuMed established a health gateway (URL: ) for laypersons, listing consumer health information sites. Before MedCIRCLE, documents had been evaluated using the DISCERN instrument [ 11 ]. COMB (URL: ) represents the medical profession of Barcelona. To this date, in the project "Web Medica Acreditada", COMB has accredited more than 300 Spanish health websites from Spain and Latin America [ 12 ]. The Knowledge Management Department of the German Research Center for Artificial Intelligence DFKI GmbH provided consultancy services especially in the area of ontology modeling. DFKI also provided the technical infrastructure and development resources for the project. CISMeF terminology The CISMeF team is composed of five medical librarians, two medical informaticians, one engineer, three Ph.D. and two Master students in Computer Science. CISMeF uses two standard tools for organizing information: the MeSH (Medical Subject Headings) thesaurus from the US National Library of Medicine, and several metadata element sets [ 13 ]: (a) 11 of 15 items of the Dublin Core metadata format to describe and index all the health resources included in CISMeF (author or creator, date, description, format, identifier, language, publisher, resource type, rights, subject and keywords, and title), (b) the 11 elements of the Educational category from Learning Object Metadata (LOM) for teaching resources, (c) specific metadata for evidence-based medicine resources (indication of the level of evidence and the method to calculate it) which also describe the health content [ 14 ], and (d) the HIDDEL metadata set (Health Information Disclosure, Description and Evaluation Language) [ 15 ]. Description of the HIDDEL language HIDDEL is a metadata language and an ontology, which enables the expression of descriptive and evaluative annotations in XML/RDF. The first version of HIDDEL was initially developed during the MedCERTAIN project (MedPICS Certification and Rating of Trustworthy Health Information on the Net, ) [ 16 ]. HIDDEL evolved from MedPICS [ 17 ], a basic rating vocabulary for medical information conforming to the Platform for Internet Content Selection (PICS) [ 18 ]. HIDDEL is used to enhance transparency of health information on the Internet. HIDDEL is based on existing quality criteria such as the Health On the Net (HON) Code of Conduct [ 2 ]. It was developed together with a quality management process model. HIDDEL can be used by information providers for self-disclosure, but also by third parties such as quality-controlled health gateways, to evaluate health information providers. It presents three levels of evaluation: (a) self-disclosure (b) evaluation by non-medical experts, and (c) evaluation by medical experts. As a quality-controlled subject gateway, CISMeF uses HIDDEL only as a third-party. The HIDDEL vocabulary can be downloaded freely from the MedCIRCLE Web site, as long as the sources are acknowledged and requests for changes or expansions are fed back to the community. At present HIDDEL is available in four languages: English, German, French and Spanish. The use of this controlled vocabulary enables automatic translation (except for free text). The heritage process was made possible because of HIDDEL's dual structure: on the one hand, Infoprovider metadata, describing to the health information provider (e.g., the name of the person responsible for the quality of the web site), and on the other hand, Sitespecific metadata devoted to one Web site evaluation (e.g., language). In CISMeF, we have applied Sitespecific metadata to each resource (mainly quality-controlled documents) from a publisher already included in the CISMeF database. The name of the person responsible for the quality of the Web site, which is one of the Infoprovider metadata, is the same for every document of the Web site. On the contrary, the language of the document, which is one of the Sitespecific metadata, may vary from one document to another. The CISMeF team implemented the whole HIDDEL structure in the CISMeF database, which involved the creation of triggers, thus ensuring automated transfer from CISMeF to HIDDEL metadata, and the creation of new forms (interface recasting) to deal with non-CISMeF metadata. Because the HIDDEL elements are optional and repeatable, CISMeF has selected a number of 70 metadata among the 305. Most of the metadata previously used in CISMeF and in particular the Dublin Core are also included in the HIDDEL language. These metadata were automatically triggered in the HIDDEL language. Interoperability The interoperability process consists of an exchange of RDF files, containing experts' annotations "written" in HIDDEL. The semantic-based Archer Annotation System deals with RDF annotations reception. Archer is a Web application that allows annotating health information Web sites using the HIDDEL vocabulary. It is a technical platform and an organizational infrastructure that can be used by consumers, health information providers, and third party rating services. The first version of Archer was implemented as a part of MedCERTAIN, and further enhanced in the course of the successor project MedCIRCLE to allow the exchange of metadata between third party rating organizations. On another ground, through its search engine Doc'CISMeF, CISMeF provides external links to Archer backend servlets, and internal links to rated sites disclosure (see Figure 1 ). Since August 2002 the CISMeF team has embedded RDF metadata (URL: ) into the generated HTML pages, making them not only machine-readable (as every HTML page is) but also machine-processable. Therefore, one of the main goals of this metadata element set was fulfilled easily: it became interoperable with other Internet services. Moreover, an RDF Scheme describing CISMeF specific metadata was created (URL: ). In a more pragmatic way, interoperability relies on a 3 steps process (see Figure 2 ): ( 1 ) RDF files generation: a Java program (RDFWriter.class) formats evaluation data according to a MedCIRCLE RDF Schema of annotations; (2) RDF files export: a Java program (RDFSender) sends RDF files to the MedCIRCLE web server using HTTP Post; (3) Reception and ID allocation: for each transmitted file, the MedCIRCLE Web server sends back an ID number that will be used to access the exported metadata. Results The CISMeF team in the MedCIRCLE consortium has evaluated and annotated the main health information providers (or publishers) included in the CISMeF database: national agencies, medical societies, universities and hospitals. CISMeF first checks the publishers' information without asking the health information provider to self-declare any metadata as described in the MedCERTAIN quality management process. CISMeF used HIDDEL to select and evaluate the 270 health publishers most represented in CISMeF and made the results of their evaluations explicit and accessible using RDF metadata. These were exported into the searchable MedCIRCLE Open Directory. Each site rated in CISMeF has a record in the CISMeF database that generates an RDF into HTML file. The search tool Doc'CISMeF (URL: ) displays the information originating from each of the publishers that were evaluated with a specific MedCIRCLE button, which is linked to the MedCIRCLE central repository where HIDDEL metadata elements are displayed (see Figure 3 ). Seal of trust, such as the one developed by HON, is a "quality seal" or a "seal of approval": i.e. the HON logo provides an accreditation, whereas the MedCIRCLE seal is not a "quality seal" but a "transparency seal": it is a button allowing health professionals and consumers to access metainformation. The presence of a MedCIRCLE button on a health Web site does not imply, in any way, that the site meets minimum required standards. This decision is left up to the user. In contrast, a seal of trust is a quality seal: i.e. every Web site with a seal of approval (such as the HON seal) has been previously accredited a third party. Nonetheless, every resource included in CISMeF and those evaluated by the MedCIRCLE process are quality-controlled. The MedCIRCLE consortium takes a very neutral approach and does not impose but strongly recommends certain procedures or minimum metadata, taking into account that collaborating gateways, accreditors, certifiers, raters may approach from very different angles. CISMeF has applied full heritage from the evaluated publishers: each document from a MedCIRCLE rated publisher, indexed in CISMeF, will also receive the MedCIRCLE button of the publisher with the same link to MedCIRCLE central repository. The idea is to keep the common Infoprovider elements for every document, and to use CISMeF metadata to disclose Sitespecific elements specific to each document, in particular the indexing with the MeSH thesaurus. At the end of the project in December 2003, starting from 270 websites, the translation from CISMeF metadata to HIDDEL, led to 6,480 evaluated resources in CISMeF in September 2003 (49.8% of the 13,012 resources included in CISMeF). All CISMeF selected HIDDEL metadata (70 out of 305) are displayed in the Doc'CISMeF record in RDF into HTML. The top five publishers indexed in CISMeF, which produced trustworthy documents in French are: Grenoble Medical School (N = 435), Health Canada (n = 275), Strasbourg Medical School (N = 263), and French Ministry of Health (N = 248). Every new document (e.g. clinical guideline or teaching material) included in CISMeF from one of the 270 main publishers evaluated through the MedCIRCLE process inherits automatically: (a) a MedCIRCLE button linking to the repository and (b) HIDDEL metadata included in CISMeF database and displayed in the Doc'CISMeF record in RDF into HTML. On the other hand, a document that comes from one of the 270 publishers evaluated within the MedCIRCLE project but not included in the CISMeF database will not receive a MedCIRCLE button in the CISMeF gateway. However, this more global transitivity could be applied in a more generic search tool such as Google. Since February 2003, when the MedCIRCLE button became operational in the CISMeF gateway, the CISMeF team has decided to go on (keep) applying the MedCIRCLE transparency process after the end of the project with the following rules: (a) check every month if there is a new publisher with five documents already included in CISMeF and (b) if so, begin the MedCIRCLE process for these publishers and apply transparency heritage for their respective documents. We applied these rules after the end of the EU-funding project. In March 2004, the CISMeF database contained a complete evaluation through the MedCIRCLE process for 346 publishers (+76, as compared to the EU-grant proposal) and 7,053 documents from these publishers (53.3% of the 13,227 resources included in CISMeF). In the MedCIRCLE repository, end-users can access an aggregate view of what people say about a certain Web site by clicking: The CISMeF gateway is one of many possible producers of trustworthy metadata regarding a health information provider. Metadata from the Open Directory can also be fed into search engines and other gateways. Discussion As the number of health related Web sites worldwide has been estimated as being around 100,000, complete coverage by a single third party evaluation body is impossible. Instead, a collaborative approach as shown in this paper has to be promoted, whereby different rating services or organizations use comparable standards and a common metadata language. More recently, the Health on the Net Foundation has developed a HON tool bar in the course of the EU-funded Active Health (Active Environment for Health Promotion and Disease Prevention, URL: ) consortium (URL: ): this HON tool bar is indicating if the site is accredited directly by HON or indirectly by one specific accredited Web sites as health gateways such as CISMeF or MedlinePlus. In this context, HON is creating a seal of quality trust heritage where MedCIRCLE is creating a seal of transparency heritage. A formal evaluation of these two examples of heritage (quality seal vs. transparency seal) is mandatory to check their respective hypothetical added value. One of the main findings of the MedCIRCLE consortium in the course of the project has been that there is no absolute objective quality of a Web site. Quality is to a certain degree subjective, may vary in time and also according to the eye of the beholder. A Web site that a consumer looking for health information finds acceptable one day may be unacceptable another day. For example, a consumer may search general information for one drug (e.g. after a TV show) and finds advice on a patient information Web site. Later, the same consumer searches for the same drug for his/her child. But this time, he/she checks if the respective Web site is sponsored by a pharmaceutical company. He/she comes to the same Web site as before, but this time this Web site will be unacceptable. The context of the search changed, making a general advice acceptable and a specific advice unacceptable. By providing metadata about health related websites, MedCIRCLE allows the health information consumers to decide themselves if a website is of good quality. Trust is improved by enhancing transparency. More globally, the MedCIRCLE consortium will lead to a safer Internet by providing a seal of transparency to over 7,000 resources on the Internet via the CISMeF search engine. The precaution principle is now widely accepted in the European Union, which develops Action Plans, such as the Action Plan for Safer Use of the Internet (URL: ), which partially granted the MedCIRCLE consortium. The MedCIRCLE button does not directly fulfill the objective to only identify reliable health information. This objective is in fact fulfilled by th HON initiative. In contrast, the MedCIRCLE consortium indirectly fulfill this objective (i.e. to identify only reliable health information) by providing a seal of transparency. The Netizen (citizen on the Internet) has the active role to evaluate the reliability when reading the HIDDEL metadata after clicking the MedCIRCLE button. With a seal of trust, the role of the Netizen is more passive but leads to a faster trust information access. Nevertheless, a drawback of the MedCIRCLE button is a possible misunderstanding by end-users, who might confuse it with a quality seal and therefore may forget to actively press the MedCIRCLE button, which may hinder their valid judgement. Here again, a formal evaluation of theses two approaches (transparency vs. trust) is necessary. While using Net Scoring, CISMeF was acting as an implicit third party. One of the main results of the MedCIRCLE project, from the CISMeF team's point of view, is that CISMeF proceeded from being an implicit to being an explicit third party thanks to the creation of the MedCIRCLE button now used for 346 publishers (2.6% of the resources included in CISMeF) and 7,053 resources (53.3% of the resources included in CISMeF), which multiplies by 20 the number of resources with a seal of trust. The HIDDEL language is totally embedded in the CISMeF metadata element sets. This allows a very easy interoperability with MedCIRCLE tools, and more specifically Archer. Semantic Web approaches already used in the MedCIRCLE project could open up new ways for educating health professionals and consumers and reaching less savvy health professionals and consumers, because part of the intelligence and knowledge currently required to critically appraise information on the health professional or consumer Web site could be built into the search tools. The feasibility of this approach has been already demonstrated by CISMeF but also by the German (AQuMed) and Spanish partners (COMB). The impact on health professionals and consumers is subject to ongoing investigation within the MedCIRCLE project. The Semantic Web may provide the health professional or the consumer with a greater capacity to determine the reliability of a given health information provider or service than the Web in its current form. Conclusion With the MedCIRCLE project and transparency heritage, CISMeF became an explicit third party. Competing interests None declared. Authors' contributions SJD had the original idea of seal transparency and drafted the manuscript. DH and TRRB developed respectively in France and Germany the programs to implement the seal transparency in CISMeF & MedCIRCLE Web sites. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521492.xml |
522825 | Dietary analysis and patterns of nutritional supplement use in normal and age-related macular disease affected subjects: a prospective cross-sectional study | Background Poor diet is thought to be a risk factor for many diseases, including age-related macular disease (ARMD), which is the leading cause of blind registration in those aged over 60 years in the developed world. The aims of this study were 1) to evaluate the dietary food intake of three subject groups: participants under the age of 50 years without ARMD (U50), participants over the age of 50 years without ARMD (O50), and participants with ARMD (AMD), and 2) to obtain information on nutritional supplement usage. Methods A prospective cross-sectional study designed in a clinical practice setting. Seventy-four participants were divided into three groups: U50; 20 participants aged < 50 years, from 21 to 40 (mean ± SD, 37.7 ± 10.1 years), O50; 27 participants aged > 50 years, from 52 to 77 (62.7 ± 6.8 years), and ARMD; 27 participants aged > 50 years with ARMD, from 55 to 79 (66.0 ± 5.8 years). Participants were issued with a three-day food diary, and were also asked to provide details of any daily nutritional supplements. The diaries were analysed using FoodBase 2000 software. Data were input by one investigator and statistically analysed using Microsoft Excel for Microsoft Windows XP software, employing unpaired t-tests. Results Group O50 consumed significantly more vitamin C (t = 3.049, p = 0.005) and significantly more fibre (t = 2.107, p = 0.041) than group U50. Group ARMD consumed significantly more protein (t = 3.487, p = 0.001) and zinc (t = 2.252, p = 0.029) than group O50. The ARMD group consumed the highest percentage of specific ocular health supplements and the U50 group consumed the most multivitamins. Conclusions We did not detect a deficiency of any specific nutrient in the diets of those with ARMD compared with age- and gender-matched controls. ARMD patients may be aware of research into use of nutritional supplementation to prevent progression of their condition. | Background Poor diet is thought to be a risk factor for many diseases [ 1 , 2 ]. One way of evaluating this risk is to carry out studies using dietary assessment techniques. Food frequency questionnaires (FFQ) have been the primary method of food self-reporting in nutritional epidemiology for the past 20 years, but it is now suggested that the ability to study associations between diet and chronic diseases may be better served by using a food diary [ 3 ]. The most accurate methods for dietary assessment are direct observation in the home, or a food history, which involves a 1–2 hour interview by a specially trained nutritionist. These methods are costly and the food diary is often used when they are not possible [ 4 ]. Age-related macular degeneration (AMD) is the leading cause of blind registration and visual disability in patients over the age of 60 years in the developed World [ 5 ]. The condition affects more than 1.75 million people in the United States, and it is expected that the demographic right-shift will lead to an increase in this number to almost 3 million by 2020 [ 6 ]. In accordance with the International Classification and Grading System for Age-Related Maculopathy (ARM), and Age-Related Macular Degeneration (AMD), these abbreviations will be used throughout [ 7 ]. The term age-related macular disease (ARMD) will be used to encompass ARM and AMD. ARM is the early stage of ARMD and is most often clinically apparent over the age of 50 years. The main symptom is increasing difficulty with fine detail discrimination. AMD is the later stage of ARMD and is categorised further in to 'dry AMD' (also known as geographic atrophy, GA), and 'wet AMD' (also known as 'neovascular', 'exudative', or 'disciform' AMD) [ 7 ]. GA is the most common form, and is estimated to be present in 15% of eyes by 80 years of age [ 8 - 11 ]. Progression is slow and legal blindness has been estimated to occur between 5 and 10 years [ 12 ]. Exudative AMD is less common, occurring in 5.2% of the population over 75 years [ 13 ], but accounts for a 90% blind registrations[ 14 ]. Patients experience rapid, significant loss of central vision as a result of growth of new blood vessels beneath the retina. The prevalence of GA and exudative AMD in the US population over 40 years of age has been estimated at 1.47% [95% confidence interval (CI), 1.38% – 1.55%] [ 6 ]. The likelihood of visual deterioration in those with exudative AMD may be reduced with laser treatment [ 15 - 18 ], although success is limited. The paucity of treatment options has prompted interest in the identification of risk factors, as well as the development of prevention strategies. The three main risk factors are increasing age [ 19 - 26 ], smoking [ 22 , 27 - 29 ], and genetic predisposition [ 30 - 34 ], although other proposed factors include gender [ 35 , 36 ], race [ 37 - 39 ], socioeconomic factors [ 21 , 40 ], cardiovascular disease [ 21 , 31 , 41 , 42 ], and poor nutrition [ 43 - 45 ]. It is thought that people with low systemic antioxidant levels may be more prone to oxidative damage of the retina and therefore, AMD [ 46 ]. Oxidation refers to removal of electrons and is mediated by reactive oxygen intermediates (ROI), which include free radicals, hydrogen peroxide, and singlet oxygen. Free radicals are molecules that contain one or more unpaired electrons in their outer orbits [ 47 ], and they extract electrons from other molecules in order to achieve stability. These molecules are rendered unstable by the interaction and a cytotoxic chain reaction results. This damage is thought to contribute to the pathogenesis of many diseases [ 1 , 2 ]. The hypothesised role of oxidation in the development of AMD has prompted research into the use of nutritional supplementation [ 48 ]. The Age-Related Eye Disease Study (AREDS) found a significant odds reduction for the development of advanced AMD with antioxidant plus zinc supplementation [ 49 ], and the Lutein Antioxidant Supplementation Trial (LAST) reported that visual function in AMD patients is improved with supplementation of lutein and lutein combined with other nutrients [ 50 ]. Lutein and its isomer zeaxanthin are carotenoids, and are synthesised in plants, algae, and bacteria. In mammalian systems they can only be obtained from the diet [ 51 ]. Their selective absorption by the retina, in particular the macula, is suggestive of a protective function, and has prompted use of the term macular pigment (MP) to describe them within the retina. Lutein and zeaxanthin are believed to protect the retina in two ways. Firstly, they act as blue-light filters. Action spectrum for blue-light induced damage shows a maximum between 400 nm and 450 nm, and this is consistent with the absorption spectrum of macular pigment [ 52 ]. Secondly, they are able to quench free radicals. Energy transfer to them quenches singlet oxygen, and they are also believed to react with peroxyl radicals that are involved with lipid peroxidation [ 53 ]. The primary aim of this study was to evaluate the dietary food intake of three subject groups: participants under the age of 50 years without ARMD (U50), participants over the age of 50 years without ARMD (O50), and participants with ARMD. The secondary aim was to obtain information on nutritional supplement usage. Methods Study design Prospective cross-sectional in a clinical practice setting. Participants Seventy-four participants gave informed consent to take part in this study, which was approved by the Institutional Human Ethics Committee. Recruitment methods included sending information to Birmingham optometrists, ophthalmologists, and a specialist centre for rehabilitation of people with sight loss, an editorial in a local newspaper, recruitment e-mails sent to the Royal National Institute for the Blind (RNIB) and all staff and students at Aston University and Aston Science Park, Birmingham, UK. For analysis the participants were divided into three groups: U50; 20 participants aged < 50 years, from 21 to 40 (mean ± SD, 37.7 ± 10.1 years), O50; 27 participants aged > 50 years, from 52 to 77 (62.7 ± 6.8 years), and ARMD; 27 participants aged > 50 years with age-related macular disease, from 55 to 79 (66.0 ± 5.8 years). All participants were part of a larger study investigating the effects of nutritional supplementation on visual function in normal and diseased eyes [ 54 ]. Chi squared analysis for gender yielded no significant difference between groups U50 and O50 [χ 2 (1) = 0.104 p = 0.305] and groups O50 and ARMD [χ 2 (1) = 3.814 p = 0.051]. The difference in age is not significant between groups O50 and ARMD (t = -1.842, p = 0.071). Exclusion criteria were the presence of an ocular condition other than ARMD and the presence of medical conditions indicating a diet in which particular foods or food groups were excluded (e.g. coeliac disease). Participants were issued with a three-day food diary with verbal and written instructions explaining that they should add to their diary every time they eat or drink, describing the food as accurately as possible and giving estimates of amounts. They were also asked to provide details of any daily nutritional supplements. The diary consisted of two week days and one weekend day. The diaries were analysed using FoodBase 2000 software (The Institute of Brain Chemistry and Human Nutrition, 166–220 Holloway Road, London N7 8DB, UK), which is a computerised nutrition database containing data on approximately 3750 foods. It can be used for recipe analysis, meal analysis, and daily or weekly analysis of menus or food intakes. Data were input by one investigator and statistically analysed using Microsoft Excel for Microsoft Windows XP software, employing unpaired t-tests. Results Dietary analysis The values for energy and nutrient intakes for all participants are shown in table 1 . Table 1 Daily mean and SD values for energy and nutrient intake. Group U50 (mean ± SD) n = 20 Group O50 (mean ± SD) n = 27 Group ARMD (mean ± SD) n = 27 Energy (kcals) 1672.30 ± 425.58 1599.78 ± 331.50 1823.37 ± 546.18 Protein (g) 71.91 ± 27.62 68.14 ± 17.08 85.25 ± 18.28 Fat (g) 65.82 ± 27.77 58.44 ± 44.27 66.73 ± 21.76 Carbohydrate (g) 203.13 ± 39.46 204.43 ± 44.27 222.05 ± 82.66 Alcohol (g) 4.31 ± 4.38 4.83 ± 8.70 6.86 ± 9.54 Fibre (g) 13.04 ± 4.60 16.21 ± 5.44 17.31 ± 5.47 Cholesterol (mg) 192.19 ± 122.99 203.04 ± 82.63 242.60 ± 72.46 Zinc (mg) 8.43 ± 2.98 8.50 ± 2.58 10.07 ± 2.45 Copper (mg) 1.10 ± 0.52 1.21 ± 0.51 1.43 ± 0.54 Selenium (μg) 62.82 ± 75.84 105.01 ± 126.05 72.01 ± 62.76 Riboflavin (mg) 9.90 ± 35.82 1.57 ± 0.43 1.77 ± 0.44 Vitamin C (mg) 79.01 ± 40.67 140.59 ± 75.23 114.91 ± 60.01 Vitamin E (mg) 6.33 ± 3.49 6.71 ± 3.13 7.87 ± 3.52 Vitamin D (μg) 3.33 ± 3.06 3.05 ± 1.91 3.76 ± 2.54 Retinol equivalents (μg) 681.25 ± 499.55 679.44 ± 237.65 825.22 ± 440.54 % energy from fat 34.20 ± 7.34 32.56 ± 4.69 32.89 ± 5.60 Comparing group U50 with group O50 Group O50 consumed significantly more vitamin C (t = 3.049, p = 0.005) and significantly more fibre (t = 2.107, p = 0.041) than group U50. The mean intakes for men and women in each group are shown in table 2 . The data has been broken down into male/female subgroups because reference nutrient intake (RNI) data can differ with gender. Table 2 Mean vitamin C and fibre daily intake for groups U50 and O50 Mean vitamin C intake (mg) Mean fibre intake (g) Women under 50 years (n = 12) 77.48 ± 42.79 12.19 ± 4.06 Women over 50 years (n = 22) 147.13 ± 75.12 16.03 ± 4.92 Men under 50 years (n = 12) 78.68 ± 36.93 14.18 ± 4.78 Men over 50 years (n = 7) 125.87 ± 78.20 15.96 ± 6.48 By tradition, investigators consider a study to be adequately powered if it has an 80% chance of detecting a significant difference when one exists. The number of study participants needed to detect a clinically important difference with acceptable power, can be calculated using the required power, the expected variability of the outcomes, and the chosen probability of masking a type 1 error [ 55 ]. Power analysis shows that 20 subjects is not sufficient to have an 80% chance of detecting a difference of 25% or more of the mean value at the 5% level of significance using the unpaired t-test for alcohol, copper, cholesterol, selenium, vitamin E, vitamin D, and retinol equivalents. In other words, for these dietary consitiuents we cannot state whether we found no difference between groups because there actually was no difference, or because the study did not have enough power to detect a difference. The study was however, powered to assess the difference in means for energy, protein, fat, carbohydrate, zinc, riboflavin, and percentage energy from fat, and no significant differences were found. Comparing group O50 with group ARMD Group ARMD consumed significantly more protein (t = 3.487, p = 0.001) and zinc (t = 2.252, p = 0.029) than group O50 (see table 3 ). Power analysis shows that 27 subjects is not sufficient to have 80% chance of detecting a difference in means of 25% at the 5% level of significance using the unpaired t-test for alcohol, copper, cholesterol, selenium, vitamin E, vitamin D, and retinol equivalents. The study was powered to assess the difference in means for energy, fat, carbohydrate, fibre, riboflavin, and percentage energy from fat, and found no significant difference. Table 3 Mean zinc and protein daily intake for groups O50 and AMD Mean zinc intake (mg) Mean protein intake (g) Women over 50 years (n = 22) 8.30 ± 2.50 66.35 ± 16.12 Men over 50 years (n = 7) 9.25 ± 2.50 71.61 ± 17.46 Women with ARMD (n = 13) 9.60 ± 2.26 78.05 ± 15.98 Men with ARMD (n = 14) 10.51 ± 2.54 91.93 ± 17.73 Baseline nutritional supplement intake The results indicate that group O50 (mean ± SD; 1.44 ± 1.79) consumes significantly more types of nutritional supplement than group U50 (0.55 ± 1.11) (t = 2.220, p = 0.032). No difference found between groups O50 and ARMD, and the study was powered to have an 80% chance of detecting a difference in means of 1 at the 10% level of significance. The percentage of supplements taken for each group is shown in figure 1 . Figure 1 Daily nutritional supplement use by group. Discussion The aim of this study was to evaluate the dietary intakes of three subject groups; U50, O50 and ARMD, as well as to obtain information on nutritional supplement usage. Participants under the age of 50 years consumed significantly less dietary vitamin C than those aged over 50 years. Supplementation data shows that 7.4 % of the O50 group take uncombined vitamin C compared with 0% of the U50 group. However, a higher percentage of the U50 group take multivitamins (33.3%) compared with the O50 group (22.2%). Vitamin C is water-soluble, is involved with several biological processes. As a reducing agent it is thought to be active in protection against heart disease. It protects LDL (low density lipoprotein) cholesterol from oxidative damage and reduces platelet aggregation [ 56 ]. By enhancing nitric oxide activity, vitamin C is potentially important in lowering blood pressure [ 57 ]. High dose supplementation with an antioxidant and zinc formulation, including vitamin C was associated with a 25% reduced risk of progression of AMD in those participants already suffering with the condition [ 49 ]. Some studies, however, have found no evidence for a beneficial role of vitamin C supplementation in ocular disease. There was no relationship between cataract prevalence and vitamin C intake in two studies [ 58 , 59 ], and no relationship between cataract extraction and vitamin C intake in a third [ 60 ]. Although the antioxidant properties of vitamin C are well known, there is no clinical evidence suggesting that supplementation with vitamin C can reduce the risk of ARMD, or other ocular conditions such as cataract and glaucoma. The RNI for men and women over the age of 18 years is 40 mg. The mean intakes of men and women in the U50 and O50 groups are all above this value (table 2 ). The higher intake values of the O50 group may be explained by their increased awareness of the benefits of a balanced diet, consumption by this group of more traditional, home prepared foods, and lower consumption of convenience foods. An increased consumption of convenience foods in the U50 group may also explain why they consume significantly less fibre than the O50 group (t = 2.107, p = 0.041). Interestingly, all three groups had a mean intake value of less than 18 g, the RNI for fibre in men and women. The ARMD group consumed significantly more dietary zinc than age- and gender- matched controls. Zinc has been investigated with regard to its potential preventative role in ARMD. The AREDS group found a suggestive reduction in the risk of progression of AMD in participants supplementing with 80 mg zinc daily. Previous randomized controlled trials (RCTs) using 200 mg zinc daily found conflicting results [ 61 , 62 ], and the positive result reported by Newsome et al (1988) should be treated with caution [ 48 ]. The higher intake by ARMD participants may be explained by their awareness of research into zinc supplementation and the condition. The RNI for women over 18 years is 7.0 mg and for men over 18 years is 9.5 mg. Our results show that the mean intakes were above RNI values for all four groups. Supplementation data shows that 11.1 % of the ARMD participants supplemented with zinc, compared with 3.7 % of the O50 group, and 0 % of U50 participants. The Food Standards Agency released a report on the safety of vitamins and minerals in May 2003 and suggested a safe upper limit of 42 mg for total daily zinc intake. Zinc supplementation over 150 mg/day has been associated with gastrointestinal side effects such as cramping and nausea, as well as lethargy and blood in the urine [ 63 ]. Our results show that the ARMD participants are most at risk of exceeding the safe upper limit as they have the highest dietary and supplemental zinc intake. The ARMD group consumed significantly more protein than O50 participants. We are not aware of any investigation into a link between protein and risk of ARMD, and table 4 shows that the mean intakes are above the RNI for both men (55.5 g) and women (46.5 g). Previous studies have found a relationship between higher dietary fat intake and risk of ARM (RR 1.6) [ 64 ], and high serum cholesterol and increased risk of exudative AMD compared with low serum cholesterol levels [relative risk (RR) 4.1] [ 40 , 65 ]. However, the NHANES I found that subjects with high cholesterol intake were less likely to develop AMD than those with lower intake [odds ratio (OR) 5.1] [ 21 ]. Our results show that ARMD participants consumed more fat and cholesterol than the O50 group, although these differences were not statistically significant. The study was underpowered for cholesterol. Research into the role of alcohol consumption in the development of AMD has produced conflicting results. Several studies have found no relation [ 40 , 66 - 70 ], but consumption of beer has been related to an increased risk of retinal pigmentation (OR 1.13) and exudative AMD (OR 1.41) [ 71 ]. Both men (RR 2.16) and women (RR 2.20) in the highest category of wine intake (2 or more glasses per day) have been shown to be at increased risk of AMD [ 67 ]. This association was strongest with white wine, and interestingly the NHANES I determined that red wine is associated with a lower risk of AMD [ 21 ]. This may be related to the antioxidant properties of the phenolic compounds within red wine [ 72 ]. Our data shows that the ARMD group consumed more alcohol than both the U50 and O50 groups, although these differences were not statistically significant and the study was underpowered for alcohol. The non-significant differences found between groups for alcohol, copper, cholesterol, selenium, vitamin E, vitamin D, and retinol equivalents may have occurred because there truly was no difference, or because the study had insufficient power to detect a difference. Because of the variability of the data, subject numbers required per group for 80 % power at the 5 % significance level are 467 for alcohol, 50 for copper, 44 for cholesterol, 341 for selenium, 59 for vitamin E, 113 for vitamin D, and 71 for retinol equivalents. Multivitamins were the most commonly taken supplement by the U50 group (30.0 %), compared with cod liver oil for the both the ARMD group (33.3 %) and O50 participants (22.2 %). Seventy-five percent of the specific ocular health related supplements were taken by the ARMD group, 25 % by the O50 group, and 0 % by the U50 group. Conclusion We did not detect a deficiency of any specific nutrient in the diets of those with ARMD compared with age- and gender-matched controls. A higher percentage of ARMD participants consume specific ocular health nutritional supplements (33.3 %) compared with age- and gender-matched controls (11.1 %) and U50 participants (0 %). The U50 group consumed a higher percentage of multivitamins, but significantly less vitamin C and fibre than the O50 group. This suggests that the younger age-group might use supplementation to ensure adequate consumption of vitamins and minerals. The ARMD group consumed more dietary zinc, more supplemental zinc, and the highest percentage of ocular health related supplements. This may suggest that information regarding the results of studies investigating the role of nutritional supplementation in reducing the risk of onset or progression of AMD is reaching patients. These results however, may be confounded by the fact that the ARMD participants used in this study were enrolled in an RCT investigating the use of nutritional supplementation in ARMD. Participants in research projects may be more aware of scientific developments and more likely to investigate their condition and potential therapies. Competing interests The authors declare that they have no competing interests. Authors' contributions HB participated in the design of the study, carried out data collection, input, and analysis, and drafted and developed the manuscript. FE participated in the design of the study and development of the manuscript. Both authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522825.xml |
532388 | Secreted Bacterial Effectors and Host-Produced Eiger/TNF Drive Death in a Salmonella-Infected Fruit Fly | Death by infection is often as much due to the host's reaction as it is to the direct result of microbial action. Here we identify genes in both the host and microbe that are involved in the pathogenesis of infection and disease in Drosophila melanogaster challenged with Salmonella enterica serovar typhimurium (S. typhimurium). We demonstrate that wild-type S. typhimurium causes a lethal systemic infection when injected into the hemocoel of D. melanogaster . Deletion of the gene encoding the secreted bacterial effector Salmonella leucine-rich (PslrP) changes an acute and lethal infection to one that is persistent and less deadly. We propose a model in which Salmonella secreted effectors stimulate the fly and thus cause an immune response that is damaging both to the bacteria and, subsequently, to the host. In support of this model, we show that mutations in the fly gene eiger, a TNF homolog, delay the lethality of Salmonella infection. These results suggest that S. typhimurium -infected flies die from a condition that resembles TNF-induced metabolic collapse in vertebrates. This idea provides us with a new model to study shock-like biology in a genetically manipulable host. In addition, it allows us to study the difference in pathways followed by a microbe when producing an acute or persistent infection. | Introduction A hallmark of pathogens is their ability to manipulate their hosts at both a cellular and organismal level. This exploitation of the host can involve the production of specific toxins, secretion of virulence effectors into host cells via type III and type IV secretion apparatuses, or mimicry of host signaling molecules ( Young et al. 2002 ; Gruenheid and Finlay 2003 ). The host organism can combat the infection with an array of innate and adaptive immune responses. Because there are so many possible combinations of thrusts, feints, and parries, there are many potential outcomes to this battle, ranging from a simple skirmish to defeat for one or the other of the combatants. As a result, microbes can produce acute and virulent infections in diseases such as cholera or plague or can take a path to cause persistent infections as in tuberculosis, Lyme disease, or host-adapted salmonellosis. It is important to understand which bacterial and host factors determine whether a microbe will cause epidemic disease, be a brief nuisance infection, or become a commensal. Salmonella enterica serovar typhimurium (S. typhimurium) is naturally infectious to mice through an oral route and causes a systemic disease resembling human typhoid fever ( Lucas and Lee 2000 ). S. typhimurium crosses the gut epithelium by entering and then killing M cells of the Peyer's patch. Once across the epithelial barrier, Salmonella infect macrophages and spread to the mesenteric lymph nodes and subsequently to other organs. S. typhimurium has two type III secretory apparatuses (TTSAs) that translocate effector proteins across both bacterial and host membranes into the host cell cytoplasm. One TTSA encoded by Salmonella pathogenicity island 1 (SPI1) plays an important role in cell entry, whereas a second TTSA, SPI2, alters the intracellular environment of the host cell to permit Salmonella growth. Salmonella likely does not find itself in the hemolymph of Drosophila in nature, but by placing it there we can ask some questions that are difficult to approach using other techniques. The fly is able to fight invading microorganisms via an innate immune response that is composed of at least three arms ( Khush and Lemaitre 2000 ). First, there is an inducible humoral immune response, which involves the secretion of antimicrobial peptides by a liver-like organ called the fat body. Second, there is a melanization response that exposes microbes to reactive oxygen as melanin is deposited on the invader. Third, there is the cellular immune response, which can result in the phagocytosis of relatively small organisms like bacteria or the encapsulation of larger parasites such as nematodes or parasitoid wasp eggs. The humoral immune functions are currently the most thoroughly characterized part of the fly's immune system. This aspect of the immune response is triggered when microbial molecules are recognized by fly pattern recognition receptors and signals are transmitted through the Toll and immune deficiency (IMD) pathways to activate three nuclear factor κB-like transcription factors, dorsal, Dorsal-related immunity factor, and Relish. It was work in this area that led to the discovery of Toll's central role in vertebrate immune recognition ( Medzhitov et al. 1997 ). Little is known about how Drosophila phagocytes affect the course of infections. It has been demonstrated that these cells act in concert with the humoral immune response to destroy invading bacteria ( Braun et al. 1998 ; Elrod-Erickson et al. 2000 ), and it is well established that they are involved in the encapsulation of parasites ( Carton and Nappi 2001 ). A potential phagocytic receptor for gram-negative bacteria as well as molecules involved in phagocytosis in cultured Drosophila cells have been identified ( Ramet et al. 2002 ). Still, we have much to learn about how the phagocytes find and phagocytose microbes, kill microbes, or send signals to the body to indicate that an infection is in progress. To focus attention on the cellular immune response, we have been characterizing the interaction between Drosophila phagocytes and pathogens that are specialized at growing within phagocytes. These bacteria have evolved methods of defeating phagocytes. By using a combination of wild-type and mutant bacteria, we can probe the function of the Drosophila phagocyte. We have shown previously that the two intracellular pathogens Mycobacterium marinum and Listeria monocytogenes can infect Drosophila phagocytes, and that some of the pathogenesis mechanisms developed by these bacteria for use in vertebrate phagocytes also function in the fly ( Dionne et al. 2003 ; Mansfield et al. 2003 ). In this work we chose to study S. typhimurium because of its well-characterized secreted effector proteins. We found that the mutation of S. typhimurium effectors led to increased fly survival but, paradoxically, also increased bacterial survival. We propose a model that suggests the immune response of the fly can be deleterious to its health. This model predicts that the fly produces damaging immune effectors. We show that the fly homolog of tumor necrosis factor (TNF), encoded by the eiger gene, is involved in this process. Flies homozygous for mutations in eiger outlive wild-type flies infected with Salmonella . Results/Discussion We attempted to infect Drosophila with S. typhimurium by feeding bacteria to flies and by injecting bacteria into the hemocoel. Flies were resistant to feeding (unpublished data), but direct injection of approximately 10,000 bacterial cells resulted in an infection that caused death in 7–9 d, while injection of sterile medium led to a mean time of death of approximately 20 d ( Figure 1 A). Figure 1 Growth of Salmonella in Drosophila melanogaster (A) Survival of wild-type flies injected with S. typhimurium. Three sets of 60 flies were injected with approximately 10,000 cfu of SL1344 ( Hoiseth and Stocker 1981 ) from an overnight 37 °C standing culture. Injected flies were incubated at 29 °C. Survival was monitored daily. Circles, S. typhimurium -injected; squares, LB-injected; triangles, uninjected. (B) Survival of immune pathway mutant flies injected with S. typhimurium . Three sets of 20 flies were injected with approximately 100,000 cfu SL1344 and incubated at 29 °C. Fly survival was monitored at 0, 12, and 24 h postinfection. Flies infected: Black, wild-type (Oregon R); gray, imd 1 /imd 1 ; white, Dif 1 /Dif 1 . (C) Salmonella growth in infected immunocompromised flies. Flies were injected with approximately 100,000 cfu SL1344 and incubated at 29 °C for the indicated times. Flies were homogenized in LB with 1% Triton X-100 to release bacteria from cells, and the homogenates were plated on LB-streptomycin plates. Only living flies were homogenized. Flies infected: Black, wild-type; gray, imd 1 /imd 1 ; white, Dif 1 /Dif 1 . (D) Effects of gentamicin on S. typhimurium growth in the fly. Wild-type flies were injected with 10,000 cfu of S. typhimurium and then incubated at 29 °C for 7 d. Flies were then injected with either a solution containing 50 nl of 1 mg/ml gentamicin (black) or water (gray). A second group of previously uninfected flies was preinjected with gentamicin or water 15 min before bacterial challenge to determine the effects of the drug on bacteria before they were phagocytosed. All flies were then incubated after gentamicin or water injection at 29 °C for 4 h. Flies were then homogenized and plated. All error bars show standard deviation. (E) Effects of S. typhimurium inoculation size on bacterial growth in the fly. Flies were injected with SL1344 over a 1,000-fold dilution range. Flies were incubated at 29 °C. Flies were homogenized immediately after injection or following 7 d of incubation before plating. Injected concentrations: Black, 0.1 optical density at 600 nm (OD 600 ); dark gray, 0.01 OD 600 ; light gray, 0.001 OD 600 ; white, 0.0001 OD 600 . The most thoroughly characterized immune response in the fly is the humoral immune response, which involves the secretion of antimicrobial peptides into the hemocoel by an organ called the fat body ( Khush and Lemaitre 2000 ). Two highly conserved signaling systems, the Toll and IMD pathways, have been shown to control the transcription of the antimicrobial peptide genes ( Lemaitre et al. 1996 , 1997 ). The IMD pathway is implicated in raising a response to gram-negative bacteria such as Salmonella . Flies homozygous for an imd mutation succumbed to Salmonella infections rapidly, dying within 12 h of infection ( Figure 1 B). Bacterial numbers exceeded 100,000,000 per fly as the flies died ( Figure 1 C), and Salmonella in dying flies were found circulating in the hemolymph (unpublished data). Circulating bacteria were not seen in wild-type flies. In contrast to imd homozygotes, flies homozygous for a mutation in Dif, a transcription factor in the Toll pathway, did not die rapidly ( Figure 1 B and 1 C). This experiment shows that mutations in the IMD but not the Toll pathway sensitize the fly to Salmonella . Further, these results suggest that the fly's humoral immune response limits the growth of free Salmonella in the hemocoel. In mammals and birds, S. typhimurium is largely an intracellular pathogen. To determine whether S. typhimurium was indeed growing in a protected intracellular niche in the fly, we measured the in vivo sensitivity of Salmonella to gentamicin, an antibiotic that does not cross host-cell plasma membranes. To determine whether gentamicin could kill S. typhimurium in flies, 50 nl of 1 mg/ml gentamicin was injected into flies to load them with the drug. S. typhimurium was injected into the flies 15 min later and incubated for 4 h to allow the antibiotic time to act. Flies were homogenized and plated on selective medium to determine Salmonella colony forming units (cfu) ( Figure 1 D). Under these conditions, bacterial loads were 100-fold lower in antibiotic-injected flies than control flies, showing that the antibiotic could kill Salmonella immediately following injection. To determine whether S. typhimurium in an established infection were protected from gentamicin killing, we injected flies that had been infected with S. typhimurium for 7 d with gentamicin or water, and incubated the flies for 4 h before homogenizing and plating them. The day 7 time point was chosen throughout this paper, as it is the latest time point we can use before most flies begin to die from the infection. This experiment showed that bacteria that had been in the fly for 7 d were protected from the antibiotic and suggests that the bacteria were located in an intracellular location. In other bacterial infection models in the fly, death of the host occurs only when bacterial numbers increase beyond 10 6 bacteria per fly. This is the case for infections both of wild-type flies with pathogens such as Pseudomonas aeruginosa and L. monocytogenes ( D'Argenio et al. 2001 ; Lau et al. 2003 ; Mansfield et al. 2003 ) and of mutant flies with nonpathogenic bacteria such as Escherichia coli ( Lemaitre et al. 1996 ; Elrod-Erickson et al. 2000 ). This is not the case for Salmonella in a wild-type fly. To determine the growth characteristics of S. typhimurium in Drosophila , bacteria were injected over a 1,000-fold dilution range; 7 d after infection, flies were homogenized and plated. At this time, bacteria were found to cover only a 5-fold range ( Figure 1 E). Bacteria injected at low densities grew to levels between 10,000 and 100,000 bacteria per fly, but those injected at higher levels did not pass this threshold. This final number is at least 1,000 fold lower than the number of bacteria found during fatal L. monocytogenes and E. coli infections in the fly. Salmonella thus reaches a population ceiling in flies, and the flies die containing relatively small numbers of bacteria. To determine the location of bacteria within the fly, we examined flies infected with S. typhimurium expressing green fluorescent protein (GFP). Bacteria carrying the plasmid containing the macrophage-inducible gene (pmig-1) ( Valdivia and Falkow 1997 ), which induces GFP expression upon entry into the phagosome, or the strain smo22 ( Vazquez-Torres et al. 1999 ), which constitutively expresses GFP (unpublished data), were injected into larvae and flies. GFP-expressing S. typhimurium could be seen within hemocytes bled from infected larvae ( Figure 2 A– 2 D). Hemocytes in adults are mostly sessile and cannot be easily removed from the fly. However, these cells can be observed through the cuticle, and clusters of these cells are found on the dorsal surface of the abdomen, along the dorsal vessel ( Elrod-Erickson et al. 2000 ; Dionne et al. 2003 ). Salmonella were found associated with these cells ( Figure 2 E– 2 H). The distribution of bacteria seen using the two GFP constructs was similar (unpublished data). This experiment suggests that, similar to the situation in mice, Salmonella are found within phagocytes. Furthermore, the Salmonella in fly hemocytes induced the expression of GFP from a Salmonella promoter normally induced upon phagocytosis by a mouse macrophage. This demonstrates that an S. typhimurium infection-related gene is induced even when the bacteria are infecting an animal at 29 °C instead of 37 °C. Figure 2 Location of S. typhimurium in the Fly (A–D) Induction of pmig-1 in Drosophila hemocytes. SL1344 carrying pmig-1 grown standing at 37 °C overnight were dried to a slide, fixed with formaldehyde and photographed using (A) differential intereference contrast (DIC) optics and (B) GFP optics. The bacteria are not highly fluorescent under these conditions. SL1344 carrying pmig-1 and grown as described above were injected into D. melanogaster larvae and the hemocytes were isolated and fixed after 24 h incubation at 29 °C. A hemocyte is shown in (C) DIC and (D) GFP optics using the same exposures as in A and B. Intensely fluorescent bacteria in the hemocytes are visible. Bar equals 5 um. (E–H) S. typhimurium growth in living flies. SL1344 carrying pmig-1 were injected into wild-type flies and incubated for 2 d at 29 °C. (E and F) Uninfected flies are compared to (G and H) infected flies with (E and G) DIC and (F and H) GFP optics. The arrowhead highlights GFP-expressing Salmonella associated with hemocytes on the dorsal side of the fly. S. typhimurium carrying mutations in a gene encoding a regulator of virulence, phosphatase P (phoP) ( Rathman et al. 1996 ), or blocking the function of either SPI1 (orgA::Tn10) ( Jones and Falkow 1994 ) or SPI2 (ssrA::miniTn5) ( Shea et al. 1996 ), were injected into flies. All of these mutants killed flies more slowly than wild-type bacteria ( Figure 3 A, graph area “i”). However, analysis of bacterial growth within infected flies revealed a more complicated story. PhoP mutants produced infections that were less lethal than wild-type Salmonella, but the infecting bacteria grew to the same levels seen in wild-type infections ( Figure 3 B). In contrast, a SPI1 or SPI2 mutant caused little death, and the bacteria grew to an average of 149,000 cfu/fly ( SPI2 mutants) instead of the average of 40,000 cfu per fly found in wild-type infections ( Figures 1 D and 3 B). Thus, the level of bacterial growth of this mutant is almost 4-fold higher than for wild-type Salmonella . This suggests that if S. typhimurium cannot manipulate Drosophila cells by secreting effectors, the survival of both pathogen and host is dramatically improved. Figure 3 Effects of Salmonella Virulence Mutations on Disease in the Fly (A) Mean time to death for infected flies. Identical quantities of S. typhimurium strains were injected into Oregon R flies and survival was monitored daily (i). Infections with slr P and rescuing construct were performed separately and are thus reported separately (ii). (B) Growth of mutant Salmonella in the fly. Approximately 10,000 cfu of each bacterial strain were injected into flies. Flies were then homogenized and plated at the time of injection (black bars) or following a 7-d incubation (white bars). All error bars show standard deviation. (C–H) Phagocytosis assays in living Drosophila . To assay the effects of Salmonella infections on phagocyte function, flies were injected with approximately 10,000 cfu of each strain of S. typhimurium . Following a 7-d incubation, the flies were first injected with FITC-labeled dead E. coli and incubated for 60 min to permit them to be phagocytosed. Trypan blue was then injected to quench the fluorescence of extracellular bacteria. The area boxed in (C) was photographed using FITC optics (D–H). The flies were injected with the following bacterial strains: (D) SL1344; (E) LB (control); (F) BJ66 (SPI1); (G) P3F4 (SPI2); (H) slrP ( Table 1 ). Table 1 Primers for Mutagenesis and Testing of S. typhimurium PSLT, plasmid Salmonella typhimurium; Sit, Salmonella iron transport; Sfb, Salmonella ferric binding; Sod, superoxide dismutase; Ssp, Salmonella secreted protein Table 1 Continued To determine which of the known TTSA effector proteins contributed to this phenotype, flies were injected with Salmonella carrying in-frame deletions of these genes as well as several others we suspected might be involved in virulence in the fly ( Figure 3 A) ( Datsenko and Wanner 2000 ). Deletions of spiC, Salmonella secreted effector A (sseA), sseB, sseC, and sseD all resembled a SPI2 knockout as expected because the proteins encoded by these genes are implicated in building the SPI2 translocation machinery ( Nikolaus et al. 2001 ; Freeman et al. 2002 ; Ruiz-Albert et al. 2003 ; Zurawski and Stein 2003 ). Deletion of the gene encoding the Salmonella leucine-rich repeat-containing protein (PSlrP) produced a phenotype similar to that of the knockout of the entire SPI2 TTSA in terms of fly death ( Figure 3 A, graph area “ii”). Wild-type virulence could be restored to the slrP mutant by expressing slrP, controlled by its native promoter, in trans on a single-copy plasmid, p. slrP ( Figure 3 A, graph area “ii”). Because SPI1 and SPI2 bacteria accumulate to higher levels than wild-type strains, in the fruit fly, we measured growth of slrP- knockout bacteria. Mutation of slrP did not significantly alter the growth of Salmonella ( Figure 3 B). These experiments suggest that slrP is a determinant of Salmonella virulence in the fruit fly, but that mutation of slrP alone is not sufficient to alter Salmonella growth. The protein encoded by slrP is suspected to be a specific effector translocated directly into the host cytoplasm. Its function there remains unknown ( Miao et al. 1999 ; Miao and Miller 2000 ; Waterman and Holden 2003 ). The results we obtain during fly infection differ from what is found in mammalian and avian hosts. SPI1 is required by Salmonella for breaching the gut barrier but is dispensable if the bacteria are introduced systemically by intraperitoneal or intravenous injection. Both phoP- and SPI2 -mutant Salmonella are highly attenuated even when injected into mammalian hosts; host survival increases, and the mutant bacteria do not readily replicate and are cleared during infections. In the fly, not only does host survival increase during infection by SPI1 or SPI2 mutants but, paradoxically, the bacteria are not cleared and replicate better than their wild-type parents. This is an interesting difference, because it separates mere bacterial presence from pathogenesis and disease. It remains to be determined why the fly does not clear the mutant bacteria in the manner that is seen in the mammalian host. The analysis of adult hemocytes in the fly is difficult, because we lack good molecular markers for them, and the cells are sessile and rare enough to make them difficult to find regularly in tissue sections. We therefore turned to a functional assay to monitor the behavior of these infected cells by determining their ability to carry out one of their definitive behaviors, phagocytosis. Flies were infected with wild-type and mutant Salmonella and then assayed for the phagocytic capacity of the hemocytes on their dorsal surface. Fluorescein isothiocyanate (FITC)-labeled dead E. coli were injected into flies and incubated for 60 min to allow time for phagocytosis to occur. Trypan blue was then injected into the flies to quench the fluorescence of extracellular bacteria. Uptake of the E. coli was then monitored by observing the flies under FITC illumination with a dissecting microscope. At day 1 post- Salmonella infection, hemocytes infected with wild-type, SPI1 -mutant, SPI2 -mutant, and SlrP bacteria showed similar phagocytic activities (unpublished data). Following 7 d of infection, flies infected with wild-type or slrP -mutant bacteria had greatly reduced numbers of phagocytic cells ( Figure 3 C– 3 H). In contrast, phagocytes remained active during the course of infection by SPI1 or SPI2 mutants. Our interpretation of this experiment is that the wild-type Salmonella infection results in death of phagocytes or a reduced capacity for phagocytosis. This reduction is not dependent on SlrP function. In mammals, cytokines such as TNF and interferon relay information about infection through the body. Cytokine expression, in particular TNF expression, is responsible directly and indirectly for a significant degree of the pathology observed during microbial infection ( Beutler and Rietschel 2003 ). Flies have one known TNF-like protein, encoded by the gene eiger ( Igaki et al. 2002 ; Moreno et al. 2002 ). Overexpression of eiger can induce cell death, but no phenotype had been identified for loss-of-function mutants thus far. We infected wild-type and eiger – flies with Salmonella to determine whether this fly TNF homolog played a role in the pathogenesis of Salmonella infections in Drosophila . The mean time to death was lengthened by 3 d in the two eiger mutants tested ( Figure 4 A). This experiment suggests that eiger /TNF signaling during a Salmonella infection contributes to the rate of death of the fly. Accumulation of Salmonella did not differ between eiger mutants and the background fly strain ( Figure 4 B). This suggests that eiger mutants do not directly affect Salmonella growth, but do markedly affect host survival. RNA transcripts of eiger were not significantly altered during Salmonella infection in comparison to Luria broth (LB)-injected controls ( Figure 4 C). This suggests that if eiger is regulated during Salmonella infection, the transcriptional changes are too small to be seen relative to expression in whole flies or the changes are posttranscriptional. Figure 4 Effects of Eiger Mutations on S. typhimurium Infections in the Fly (A) Survival of eiger -mutant flies infected with S. typhimurium . Three sets of 20 flies were infected with 10,000 cfu of SL1344 and incubated at 29 °C. Two eiger mutants ( eiger 1 and eiger 3 ) and the background strain ( w 118 ) were assayed. Survival was monitored daily. Circle, eiger 1 /eiger 1 ; square, eiger 3 /eiger 3 ; triangle, w 118 . Solid shapes indicate LB-injected flies; open shapes indicate SL1344-injected flies. Mantel-Cox analysis demonstrated p < 0.001 when comparing infected wild-type to eiger -mutant flies. (B) Growth of S. typhimurium in eiger -mutant flies. Flies were infected with 10,000 cfu of SL1344 and plated at the time of injection or following a 7-d incubation. Black, time = 0; white, time = 7 d. All error bars show standard deviation. (C) Effects of Salmonella infection on eiger RNA transcript levels. Total RNA was extracted from five flies per sample on days 0, 1, 3, 5, and 7 postinjection with (open circles) SL1344 or (closed squares) LB. Quantitative real-time RT-PCR was performed. Relative eiger transcript quantity is expressed as the fold-difference in comparison to the day 0 value. All error bars show the standard deviation of three RNA preparations. In human disease, morbidity and mortality are often the result of immune responses to invading pathogens rather than to direct action of the pathogens themselves ( Beutler and Rietschel 2003 ). Fever, inflammation, and shock are examples of such processes. In plants, a model is emerging in which host cells monitor important cell functions and respond to their perturbations by pathogens ( Staskawicz et al. 2001 ; Schneider 2002 ). We suggest the infection caused by Salmonella in the fly has attributes of both models. Salmonella growth appears to be restricted to hemocytes by action of the humoral immune response. Perhaps the manipulation of these hemocytes by secreted effectors induces additional immune responses that limit bacterial growth. This resembles what is seen in a resistant plant infected by a bacterium expressing the appropriate avirulence protein. Such proteins are secreted into the plant cell's cytoplasm via a TTSA, and they alter the host cell's physiology, presumably to make growth conditions for the bacteria more favorable. Resistant plants can sense this physiological change and raise a type of immune reaction called a hypersensitive response. In the case of Drosophila, we suggest that the fly's immune response not only limits the growth of the pathogen but also damages the fly and ultimately leads to its death. When S. typhimurium is prevented from using its secreted effectors, the bacteria survive in the phagocyte and grow to higher numbers, possibly because the bacteria are not being attacked as intensely by the host. The result is that there are more bacteria in the fly and host death is greatly delayed. These two properties appear to be separable. The mutations in eiger suggest that there are signaling events that increase the death rate in the fly but do not alter the numbers of Salmonella . These experiments provide a new genetic model to explore microbial choice of pathogenic lifestyles in addition to a potential new genetic model for the study of TNF-induced metabolic collapse. Materials and Methods Injection assays Bacteria and medium were injected in a volume of 50 nl through a pulled glass needle. The injection volume was regulated using a Picospritzer III injector (Parker Hannifin, Rohnert Park, California, United States). The needle was placed in the anterior abdomen on the ventrolateral surface. One-week-old male flies were used for all experiments. All experiments were performed in triplicate with at least 20 flies in each replicate. Oregon R flies were used as our wild-type strain. Determination of CFUs in flies Infected flies were homogenized in LB containing 1% Triton X-100. Three infected flies were homogenized together either with a small pestle or by shaking with 0.4 mm glass beads. Diluted homogenates were plated on LB-agar with 50 μg/ml streptomycin. In vivo phagocytosis assay This assay was performed essentially as described previously ( Elrod-Erickson et al. 2000 ). Infected flies were injected with 50 nl of 1 mg/ml FITC-labeled dead E. coli (Molecular Probes, Eugene, Oregon, United States) and incubated for 1 h at 25 °C to permit phagocytosis of the bacteria. Trypan blue (4%) was then injected into the flies. Enough dye was injected to turn the entire fly blue. Flies were observed under a Leica MZ3 fluorescent dissecting microscope (Leica, Wetzlar, Germany) using GFP epifluorescence optics, and photographed with an ORCA camera (Hamamatsu, Osaka, Japan) using Openlab software (Improvision, Coventry, UK). Generation of Salmonella knockouts and slrP complementation plasmid Isogenic gene knockouts were made in Salmonella strain 14028s/pKD46 ( Datsenko and Wanner 2000 ). Transformants were verified by PCR using primers with homology to flanking regions of the target gene. The deleted gene region was transferred to our test strain, SL1344, using standard P22 lambda phage transduction. Transductants were selected on LB agar containing kanamycin and 10 mM EGTA overnight at 37 °C, and mutations were verified by PCR. Primer information is provided in Table 1 . Primers designated “Salmonella typhimurium (STM)# 5′ + P1” and “STM# 3′ + P4” were used to generate the kanamycin insert specific to the target gene, using pKD13 as the template. Primers designated “STM# 5′ PCR” and “STM# 3′ PCR” were used to verify recombinants. The bacterial strains and plasmids used for cloning are listed in Table 2 . The slrP complementation plasmid, p. slrP was constructed using the single copy plasmid pDM.2002 ( Detweiler et al. 2003 ). SlrP along with its native promoter was amplified from SL1344 genomic DNA using the following primers: 5′-
CGCGGATCCAGCGTTGCAGCAGAAAAT-3′ (slrP 5′ BamHI) and 5′-
CGCGGATCCTGGGTTAAGCCCGTTTAC-3′ (slrP 3′ BamHI) ( Miao and Miller 2000 ). Table 2 S. typhimurium Strains and Plasmids Mig, macrophage-inducible gene; org, oxygen-related protein; rps, ribosomal protein subunit; Tn, transposon Eiger RNA quantitation Total RNA was extracted from five flies per sample using a Qiagen RNeasy Kit (Qiagen, Valencia, California, United States). Quantitative real-time RT-PCR was performed with rT th polymerase (Applied Biosystems, Foster City, California, United States) and the following eiger primers: 5′-
GATGGTCTGGATTCCATTGC-3′ (5′ oligo), 5′-
TAGTCTGCGCCAACATCATC-3′ (3′ oligo) and 5′-6FAM-
GACGACGAGGACGACGACGTTAGCT-TAMRA-3′ (hybridization oligo). Concentrations of eiger transcripts were normalized to the expression of the D. melanogaster ribosomal protein 15a transcript in each sample ( Schneider and Shahabuddin 2000 ). All experiments were performed in triplicate. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC532388.xml |
522824 | Evaluation of the Taguchi methods for the simultaneous assessment of the effects of multiple variables in the tumour microenvironment | Background The control of proliferation, differentiation and survival of normal and malignant cells in the tumour microenvironment is under the control of a wide range of different factors, including cell:cell interactions, cytokines, growth factors and hormonal influences. However, the ways in which these factors interact are poorly understood. In order to compare the effects of multiple variables, experimental design becomes complex and difficult to manage. We have therefore evaluated the use of a novel approach to multifactorial experimental design, the Taguchi methods, to approach this problem. Method The Taguchi methods are widely used by quality engineering scientists to compare the effects of multiple variables, together with their interactions, with a simple and manageable experimental design. In order to evaluate these methods, we have used a simple and robust system to compare a traditional experimental design with the Taguchi Methods. The effect of G-CSF, GM-CSF, IL3 and M-CSF on daunorubicin mediated cytotoxicity in K562 cells was measured using the MTT assay. Results Both methods demonstrated that the same combination of growth factors at the same concentrations minimised daunorubicin cytotoxicity in this assay. Conclusions These findings demonstrate that Taguchi methods may be a valuable tool for the investigation of the interactions of multiple variables in the tumour microenvironment. | Introduction The control of proliferation, differentiation and survival of normal and malignant cells is under the control of a wide range of different factors. These include cell:cell interactions, immune regulatory factors, hormonal influences, and local environmental influences. However, the way in which these factors interact to regulate the dynamics of the malignant cell population are poorly understood. It is important to identify important factors and the way that they interact in order to rationalise treatment and develop new therapeutic options. However, one of the main problems is the difficulty in designing experiments to compare the effects and interactions of multiple variables. For example, a traditional experimental design to compare seven independent variables at three different concentrations each requires a large number of individual experiments (2187 experiments). The logistical and resource implications of this experimental design make these experiments very difficult to carry out. We have investigated the use of an alternative approach to experimental design, the Taguchi Methods [ 1 ]. Taguchi methods use orthogonal array distribution to design an experiment producing smaller, less costly experiments that have a high rate of reproducibility. A study involving 7 factors at 3 different concentrations can be conducted with only 18 individual experiments. Besides being efficient, the procedures for using Taguchi designs and methods are straightforward and easy to use. These methods have previously been used in PCR optimisation [ 2 , 3 ], baculovirus expression [ 4 ], ball and socket prosthesis design for total hip replacement surgical procedure [ 5 ], ELISA optimisation [ 6 ], and also in the evaluation of medical diagnostic tests [ 7 , 8 ]. We have therefore used a simple and reproducible assay, the MTT assay, to evaluate whether the Taguchi methods can be used to investigate the effect of G-CSF, GM-CSF, IL3 and M-CSF on daunorubicin mediated cytotoxicity in K562 cells. Taguchi Methods Taguchi methods consist of 3 phases: designing the experiment, running and analysing, and confirming and validating the assumptions. After selecting the variables to be studied, Taguchi methods depend on distributing the factors under study in an orthogonal array, which distributes the variables (factors) in a balanced manner. Examining a typical orthogonal array (Table 1 ), where each factor has 2 levels or concentrations, reveals that each level has an equal number of occurrences within each column. For each column of the orthogonal distribution below, level 1 occurs four times, and level 2 occurs four times as well [ 1 ]. This idea of balance goes farther than meaning simply an equal number of levels within each column. The relationship between one column and another is arranged so that for each level within one column, each level within any other column occurs an equal number of times as well. With reference to Table 1 , it can be observed that factor A is assigned to column 1, and for A at level 1, factor B is at level 1 twice and at level 2 twice. The same is true for factor A at level 2. Looking at the last column, the same relationship between factors A and G is also noted. No matter which two columns are selected, the same will be true. The ramifications of this orthogonality among columns are the basis of the statistical independence of orthogonal arrays; hence the effect of each factor can be separated from the others. Therefore, an estimation of the effect of any one particular factor tends to be accurate and reproducible because the estimated effect does not include the influence of other factors. Furthermore, each factor can be assigned a significance weight to denote its importance in affecting the end result of the experiment. Table 1 Orthogonality. The relationship between one column and another is arranged so that for each level within one column, each level within any other column occurs an equal number of times as well. Factor A, at level 1 occurs 4 times and at level 2 occurs 4 times as well. This equal occurrence is true for all factors involved in any orthogonal array. A B C D E F G Results 1 1 1 1 1 1 1 1 Y1 2 1 1 1 2 2 2 2 Y2 3 1 2 2 1 1 2 2 Y3 4 1 2 2 2 2 1 1 Y4 5 2 1 2 1 2 1 2 Y5 6 2 1 2 2 1 2 1 Y6 7 2 2 1 1 2 2 1 Y7 8 2 2 1 2 1 1 2 Y8 Each array can be identified by the form L A (B C ), the subscript L, which is designated by A , represents the number of experiments that would be conducted using this design, B denotes the number of levels or concentrations within each column which denotes how many levels or concentrations could be investigated, while the letter C identifies the number of columns available within the orthogonal array which indicates how many factors or variables could be included in the experiment [ 1 ]. For example the orthogonal array L 8 (2 7 ) means that 8 experimental runs are needed to investigate 7 different factors, each of which is set at 2 predetermined levels or concentrations (Table 1 ). The statistical independence of these arrays enables the effect of each factor to be separated from the others, the effects to be accurate and reproducible because the estimated effect does not include the effects of other factors and the interactions between these factors to be determined. Level average analysis, as described by Taguchi [ 1 ] is one of the techniques used to explore the results of the Taguchi methods. The name derives from determining the average effect of each factor on the outcome of the experiment. The goal is to identify those factors that have the strongest effects and whether they exert their effect independently or through interacting with other factors. The equation below illustrates the method of calculating the average effect of the experiment where Y1 is the result of the first experiment, Y2 is the result of the second experiment...etc, T is the overall average of the experiment, and n is the number of the experimental runs. For example, in order to calculate the effect of the two concentrations of factor A, which are denoted A1 and A2, where A1 is the average effect of factor A at concentration 1, A2 is the average effect of the same factor at concentration 2. The relative impact of each factor (ΔX) is simply the range, which could be calculated as the difference between the highest and lowest average response of each level. For example the impact of factor A on the experiment outcome is the difference between A1 & A2. (Known statistically as the range (Δ)). The effects of all factors are calculated in the same way, then arranged in a response table, and examined for those factors with the strongest effect (i.e. highest difference Δ), in order to separate them from the weak effects. The breaking point between the strong and weak effects is identified as a change in the pattern of the difference between the ranges around the median. Besides determining the effects of the individual factors, the same technique is used to determine the strength of the impact of interactions on the product of the experiment. The calculations are performed as the previous section. In order to determine the interactions between A and B, the average result of each 2 factors combined must be determined. This is achieved through calculating the values of 4 points: A1B1, A1B2, A2B1, and A2B2, where A1B1 is the average result generated due to the interaction between concentration 1 of both factors, A1B2 is the result of the interaction between concentration 1 of factor A and concentration 2 of factor B, A2B1 represents the interaction between factor A at concentration 2 and factor B at concentration 1, while the fourth point A2B2 is the interaction between both factors at concentration 2. These 4 points are then presented graphically to show the strength or weakness of the interaction. Whether the interaction is weak, mild, or strong depends on whether the two response lines are parallel, converging or intersecting, with intersecting lines indicate a strong interaction, and parallel lines indicate no interactions. Once the strong factors and interactions have been identified, an estimate of their combined effect is calculated and the new experiment is designed according to these assumptions. An experiment is then carried out – referred here to as "confirmation run"- to validate the assumptions upon which the new experiment was based. Conducting a confirmation run and the comparison between the actual and the predicted results is necessary. If however, the confirmation results are disappointing, the planning phase must be re-evaluated and the elements that went into the experiment must be reviewed. A possible cause could be the omission of a key factor from the experiment, for example a powerful interaction was not considered. Another common cause is the setting of factor levels too close together for the experiment. In these situations, the factor is found insignificant during the analysis and is not accounted for in the validation. The confirmation run should include the best or preferred settings for mild and weak influences as well as the strong ones. However, the less influential factors are not incorporated into the prediction equation. The reasoning is that the differences in the average results may be due to experimental variation, and to incorporate their effects could result in an overestimate of the predicted results. This could lead to a disappointing confirmation run when actually the results would have validated the experiment analysis if the predicted results had not been artificially high or low. Methods Cell Culture K562 cells were cultured in RPMI-1640 medium supplemented by 10% (v/v) foetal bovine serum, 50 μg/ml penicillin and 25 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO 2 -95% air. Cells were plated in 96 well microtiter plates (200 μl) at a density of 3 × 10 4 cells/ml. Cells were co-cultured in the presence of 0.1 μg/ml daunorubicin. Cytokines In all experiments K562 cells were co-incubated in the presence or absence of cytokines concentrations shown in Table 2 . All cytokines were purchased from R&D Systems, UK. Table 2 Concentrations of cytokines used. 1 2 A MCSF 100 U/ml 300 U/ml B IL3 10 ng/ml 50 ng/ml C GMCSF 10 ng/ml 50 ng/ml D GCSF 10/ ng/ml 50 ng/ml MTT Assay 50 μl of MTT (3–4,5-dimethylthiazol 2,5-diphenyl tetrazolium bromide) (5 mg/ml) was then added to each well and incubated at 37°C for 4 hours. The resulting deep blue crystals were dissolved in 0.04 N HCl Isopropyl alcohol, and the absorbance measured using a scanning multiwell spectrophotometer at dual wavelength 570–630 nm. All measurements were performed in triplicates. The % survival was calculated as Classical Experimental Design In classical experimental design the effect of each factor, each concentration and each interaction is tested independently. In order to investigate the full interactions between 4 factors each at 2 concentrations, requires 81 individual experimental conditions to be performed. This study used 49 combinations. The structure of the 49 experiments is shown in Table 3 , where runs 1–16 were designed to include all the different possible combinations of all 4 cytokines together. For example, experimental run 3 was carried out after adding 100 U/ml MCSF, 50 ng/ml of both IL-3 and GMCSF, and 10 ng/ml GCSF to the medium. Runs 17–24 included the effects of each cytokine individually; two concentrations of each cytokine were tested. For example in run 17,, only MCSF was added to the medium at concentration 1 (100 U/ml), while in run 18 the same cytokine was added at concentration 2 (300 U/ml). Runs 25–48 were planned to included the different possible interactions between each 2 cytokines, for example in run 25 both MCSF (100 U/ml) and IL-3 (10 ng/ml) were added to the medium, in run 26 MCSF (100 U/ml) and IL-3 (50 ng/ml) were added, in run 27 MCSF (300 U/ml) and IL-3 (10 ng/ml) were added, and in run 28 MCSF (300 U/ml) and IL-3(50 ng/ml) were added. Finally, experimental run 49 was carried out without adding any cytokines to the medium. Table 3 The whole set of the 49 experiments carried out. Runs 1–16 included all possible combinations of all cytokines together (see text above). In runs 17–24 individual cytokine were added to the medium, two concentrations of each cytokine was tested. For example in run 17 MCSF was added to the medium at concentration 1 (100 U/ml), while in run 18 the same cytokine was added at concentration 2 (300 U/ml). Runs 25 – 48 included the different possible interactions between each 2 cytokines, for example in run 25 both MCSF (100 U/ml) and IL-3 (10 ng/ml) were added, in run 26 MCSF (100 U/ml) and IL-3 (50 ng/ml) were added, in run 27 MCSF (300 U/ml) and IL-3 (10 ng/ml) were added, and in run 28 MCSF (300 U/ml) and IL-3 (50 ng/ml) were added. Experimental run 49 was carried out without adding any cytokines to the medium. All experimental runs were done in triplicate and repeated three times. A MCSF B IL-3 C GMCSF D GCSF 1 1 1 1 1 2 1 1 2 2 3 1 2 1 2 4 1 2 2 1 5 2 1 1 2 6 2 1 2 1 7 2 2 1 1 8 2 2 2 2 9 1 1 1 2 10 1 2 1 1 11 1 1 2 1 12 1 2 2 2 13 2 1 1 1 14 2 2 2 1 15 2 1 2 2 16 2 2 1 2 17 1 0 0 0 18 2 0 0 0 19 0 1 0 0 20 0 2 0 0 21 0 0 1 0 22 0 0 2 0 23 0 0 0 1 24 0 0 0 2 25 1 1 0 0 26 1 2 0 0 27 2 1 0 0 28 2 2 0 0 29 1 0 1 0 30 1 0 2 0 31 2 0 1 0 32 2 0 2 0 33 1 0 0 1 34 1 0 0 2 35 2 0 0 1 36 2 0 0 2 37 0 1 1 0 38 0 1 2 0 39 0 2 1 0 40 0 2 2 0 41 0 1 0 1 42 0 1 0 2 43 0 2 0 1 44 0 2 0 2 45 0 0 1 1 46 0 0 1 2 47 0 0 2 1 48 0 0 2 2 49 0 0 0 0 Taguchi Design L 8 (2 7 ) In order to evaluate the performance of the Taguchi methods, eight experimental runs were carried out employing the orthogonal array L 8 (2 7 ) to investigate the effect of 4 cytokines on the survival of K562 leukaemic cells. This array accommodated 4 factors MCSF, IL-3, GMCSF and GCSF. The interaction between MCSF and the other cytokines was inserted into the array. As the 3 rd column was used to examine the interaction between MCSF and IL-3, columns 5 and 6 were used to assess the interaction between the same cytokine and GMCSF and GCSF respectively (Table 4 ). All factors in this design were set at 10 and 50 ng/ml except MCSF, which was set at 100 and 300 U/ml. Table 4 Taguchi method L 8 (2 7 ). This array accommodated 4 different factors (MCSF, IL-3, GMCSF, and GCSF) each at 2 different concentrations (see above). 8 experimental runs were carried out according to the combination of factors in the array, for example, in experimental run 1 the MTT assay was carried out after mixing the cells with 100 U/ml MCSF, 10 ng/ml IL-3, 10 ng/ml GMCSF, and 10 ng/ml GCSF. The interaction between MCSF and the other three factors (IL-3, GMCSF and GCSF) was studied in this array. A MCSF B IL3 AxB C GMCSF AxC AxD D GCSF 1 1 1 1 1 1 1 1 2 1 1 1 2 2 2 2 3 1 2 2 1 1 2 2 4 1 2 2 2 2 1 1 5 2 1 2 1 1 1 2 6 2 1 2 2 2 2 1 7 2 2 1 1 1 2 1 8 2 2 1 2 2 1 2 Results and Discussion Results of the classical design The results of the 49 experimental conditions are shown in Figure 1 . Columns 1–24 represent the simultaneous combinations of the 4 cytokines. The toxicity of daunorubicin was maximally enhanced by the addition of the four cytokines to the medium i.e. MCSF at a concentration of 300 U/ml, IL-3 at a concentration of 50 ng/ml, GMCSF at a concentration of 50 ng/ml, and GCSF at a concentration of 50 ng/ml. This resulted in a highly significant reduction of malignant cell survival, from 69% (the survival rate for the control cells) to 39% (P <0.001). Figure 1 Experimental runs 1–16 show the different survival rates of K562 cells as a result of culturing the cells in medium enriched by different combinations of the 4 cytokines (GMCSF, MCSF, IL-3, and GCSF). The maximum cytotoxicity of daunorubicin was observed as a result of the addition of 300 U/ml of MCSF, and 50 ng/ml of the other 3 cytokines (experimental run 3). The maximum survival of the cells was observed when the concentration of GMCSF in this mixture was reduced to 10 ng/ml (experimental run 16). Experiments 17 – 48 suggested that MCSF interacts with the 3 other factors to affect daunorubicin cytotoxicity. This could be seen by comparing the effect of the individual factors (runs 17 – 24) with the effects of the addition of two factors simultaneously. For example, experimental run 30 shows the concurrent effect of both MCSF (100 U/ml) and GMCSF (50 ng/ml) that resulted in a survival that was significantly higher than that caused by any of the two factors alone (runs 17, 18, 21 & 22). Experimental run 26 also represents the combined effects of MCSF (100 U/ml) and IL-3 (50 ng/ml), which resulted in a survival that was higher than the resulting survival of any of the two factors individually. Run 36; on the other hand, represents the increase in daunorubicin cytotoxicity as a result of the simultaneous addition of MCSF (300 U/ml) and GCSF (50 ng/ml). All these experimental runs were done in triplicate and repeated 3 times, the results are expressed as mean ± SE. The survival of K562 cells, using the 4 cytokines simultaneously was maximally enhanced by the addition of 300 U/ml MCSF, 50 ng/ml IL-3, 50 ng/ml GMCSF, and 50 ng/ml GCSF. A significant improvement in cell survival from 69% to 76% (P 0.02) was observed Taguchi analysis The results of the 8 experiments of Taguchi's L 8 series (Table 5 ), were analysed in order to determine the mean effect of each factor. Table 5 the results of L 8 (2 7 ). Each experimental run was done in triplicate and repeated 3 times, the mean values were calculated and the results were expressed as mean ± SE. Y1 (experimental run 1), for example, = the mean survival of the cells at 100 U/ml MCSF, 10 ng/ml IL-3, 10 ng/ml GMCSF, and 10 ng/ml GCSF. The overall average of the experiment (T) was calculated as the mean of all eight experimental runs. % survival Y1 60.71 ± 5.9 Y2 67.83 ± 1.9 Y3 64.01 ± 1.1 Y4 63.51 ± 2.0 Y5 62.97 ± 4.3 Y6 72.27 ± 4.3 Y7 62.86 ± 1.1 Y8 39.84 ± 1.9 T 61.75 For example the mean effect of MCSF when added to the medium at a concentration of 100 U/ml was computed as follows: When MCSF was added to the medium at a concentration of 300 U/ml the mean effect was: These computed values were used to construct a response table (Table 6 ). This shows the average mean effect for each factor and the relative impact or range of each factor on the variability of the mean. This showed that the following concentrations were associated with lower survival of the malignant cells; 300 U/ml MCSF was associated with the survival of 59.4%, 50 ng/ml of IL-3 was associated with 57.5 % survival rate, 50 ng/m GMCSF resulted in the survival of 60.8% of the cells, and finally 50 ng/ml of GCSF was associated with 58.6% survival rate. Table 6 Response table for the orthogonal array L 8 (2 7 ). The average effect of each factor level is calculated and the range of effect of each factor is calculated as the difference between the two readings. The range of MCSF effect, for example = 64.02-59.48 = 4.53, the higher the range the stronger the effect of the factor. In this experiment the interaction between MCSF and GCSF had the strongest effect on the survival of cells. A = MCSF B = IL3 AxB C = GMCSF AxC AxD D = GCSF 1 64.02% 65.95% 57.81% 62.64% 59.21% 56.76% 64.84% 2 59.48% 57.55% 65.69% 60.86% 64.29% 66.74% 58.66% Δ 4.53 8.39 7.88 1.77 5.08 9.98 6.17 2 3 1 4 In order to determine the strong effects and separate them from the weak ones, the response table was rearranged by ranking the factors in order from the largest difference to the smallest as it can be seen in Table 7 . In this study the interaction between MCSF and GCSF (AXD) has the greatest effect on the survival of K562 cells. IL-3 (B) is next with a difference between the Δ's of 1.594. The interaction between MCSF and IL-3 (AXB) was next followed by GCSF (D). The difference between ΔB and ΔAXB is 0.507 (8.391-7.884 = 0.507), if we continue farther to factor D the difference in effects jumps to 1.711 (7.884-6.173 = 1.711). Therefore this point would be considered as the breaking point. The factors to its left (AXD, B, and AXB) are the important factors i.e. MCSF, IL-3, and GCSF. MCSF exerts strong effects through interacting with both IL-3 and GCSF. Table 7 Descending rearrangement of the response table according to strong and weak effects. The response table was rearranged according to the Δs, and the difference between the Δs was calculated and then scanned to determine the break point, which was identified as a change in the pattern of the difference between the Δs around the median. The strong factors would be on the left hand side of the break point, marked in this table in bold. AxD B = IL3 AxB D = GCSF AxC A = MCSF C = GMCSF 9.985% 8.391% 7.884% 6.173% 5.088% 4.533% 1.774% 1.594 0.507 1.711 1.085 0.555 2.759 In order to study each interaction incorporated into this design, an interaction matrix for each interaction was constructed as described above; hence a 2 × 2 matrix was constructed for each interaction. For example to construct an interaction matrix for MCSF and GCSF (AXD), four points were computed A1D1, A1D2, A2D1, and A2D2 (Table 8 ). Table 8 Interaction matrix AxD. The average effect of the four points of this interaction matrix on the survival of K562 cells. The preferred setting of this interaction that would maximise the cytotoxicity of daunorubicin is A2D2 i.e. 300 U/ml of MCSF and 50 ng/ml of GCSF. This combination would result in a survival of 51.67% of the cells. D1 D2 A1 62.11% 65.92% A2 67.56% 51.67% A1D1 is the average result of the combined effect of MCSF at a concentration 100 U/ml and GCSF at a concentration of 10 ng/ml. The interaction matrix AXD was then represented graphically (Fig 2 ) which showed intersecting lines indicating a strong interaction. Both table 8 and fig 2 were further studied to decide which combination suites the desired outcome of the experiment; hence for the smaller the better outcome, it is clear that 51.67% is the lowest survival value in this matrix (Table 8 ), i.e. A2D2 (MCSF at a concentration of 300 U/ml and GCSF at a concentration of 50 ng/ml) is associated with the lowest survival rate of the malignant cells. The interaction between MCSF and the two other factors was further studied and the preferred combinations for both interactions were A2B2 i.e. 300 U/ml MCSF and 50 ng/ml IL-3, which was associated with 51.35% survival rate, and A2C2, i.e. MCSF at concentration 2 (300 U/ml) and 50 ng/ml GMCSF, which was associated with 56.05% survival. The preferred settings suggested by this analysis to optimise the cytotoxicity of daunorubicin was combining the following factors A2B2C2D2 i.e. MCSF at a concentration of 300 U/ml, IL-3 at a concentration of 50 ng/ml GMCSF at a concentration of 50 ng/ml, and GCSF at a concentration of 50 ng/ml. Figure 2 Graphical presentation of interaction between AxD (MCSF and GCSF). Intersecting lines of this graph indicate strong interaction. A2D2 is the preferred point on the graph i.e. the combination of these two factors to produce maximum daunorubicin cytotoxicity. An estimate of the predicted response (μ) based on the selected levels was then computed. The calculations were based on the overall average value (T) and the effect that each of the recommended levels of the strong factors and interactions has on the overall average. μ = T+(A2D2 - T)+(B2 - T)+(A2C2 - T)+(A2B2 - T)-(A2 - T)-(A2 - T)-(A2 - T)-(B2 -T)-(C2-T). The reason for subtracting the individual effects of factors A, B, and C from the effects of A2B2 is that A2B2 is comprised of the effects of factor A, factor B and the interaction itself. Unless the effects of the two factors are subtracted these strong effects would be included twice and resulting in an overestimation of the predicted result. The predicted survival derived from the above prediction equation was 43%. A confirmation run that produces a %survival close to 43%would validate the assumptions of this Taguchi method. The actual confirmation run, in fact, resulted in 39.84% survival rate indicating the success of the Taguchi experiment. Further calculations were performed to determine whether the outcome of other combinations could be predicted from the Taguchi experiment and confirmed by analysis. The results are shown in Table 9 and show a close approximation in each case. Table 9 Further comparison of the predicted values from the Taguchi Methods, and the result produced by experimental analysis. Prediction Analysis Experimental run 9 66.10% 70.35% Experimental run 10 71.93% 74.38% Experimental run 11 66.14% 63.36% Experimental run 12 67.65% 65.36% Experimental run 13 48.90% 45.69% Experimental run 14 51.46% 55.22% Experimental run 15 63.65% 74.88% Experimental run 16 72.19% 76.74% Conclusion The aim of this study was to evaluate the ability of the Taguchi methods to investigate the effects of several factors simultaneously on the death and/or survival of the malignant cells, and to compare this strategy against a traditional full experimental design. A major finding of the study was that the Taguchi methods predicted the combination of factors that results in the lowest survival of the malignant cells. This agreed with the conclusions of the full experimental design but required only eight individual experiments to pinpoint this combination. However, it must be stressed that the Taguchi methods are not intended to be a replacement for traditional experimental design, but if used as a complimentary strategy can make analysis of complex interactions feasible and practical. In this study, for example, eight individual experiments produced a testable combination which required 49 individual experiments to produce in the traditional experimental design. In more complex systems, only Taguchi methods become feasible. For example, to study 13 factors at 3 different combinations would require 1,594,323 individual experiments at a cost for re agents alone of over 27 million pounds. If Taguchi methods are used, this can be reduced to just 27 individual experiments at a cost of under 500 pounds. We have described a novel experimental approach to studying the interactions of several factors on the cytotoxicity of malignant cells. We show that the method is effective in the determination of the optimum conditions, even in the presence of multiple interactions. We anticipate that this experimental strategy will have many applications in the investigation of complex interactions. For example we have used this strategy to model the complex testicular microenvironment and the ability to support the survival of acute lymphoblastic leukaemia cells (manuscript in preparation). These sort of interactions are common in the survival of malignant cells in vivo , and we propose that the Taguchi methods may be a useful strategy to understand these interactions in vitro , and to help devise and implement new therapeutic strategies. Competing interests None declared. Authors' contributions HM designed the Taguchi assay, carried out cell survival assays, and drafted the manuscript. KLY participated in the design and coordination, and produced the final manuscript. APJ conceived the study and participated in its design and coordination. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522824.xml |
551613 | Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling | Background The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of G i -coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization. Results Removal of phosphorylation sites in the C-terminus of the RASSL resulted in a mutant that was resistant to internalization and less prone to desensitization. Replacement of the C-terminus of the RASSL with the corresponding portion of the mu opioid receptor eliminated the induction of AC superactivation, without disrupting agonist-induced desensitization or internalization. Surprisingly, removal of phosphorylation sites from this chimera resulted in a receptor that is constitutively internalized, even in the absence of agonist. However, the receptor still signals and desensitizes in response to agonist, indicating normal G-protein coupling and partial membrane expression. Conclusions These studies reveal that internalization, desensitization and adenylyl cyclase superactivation, all processes that decrease chronic G i -receptor signals, are independently regulated. Furthermore, specific mutations can radically alter superactivation or internalization without affecting the efficacy of acute G i signaling. These mutant RASSLs will be useful for further elucidating the temporal dynamics of the signaling of G protein-coupled receptors in vitro and in vivo . | Background The specificity, diversity, and physiological importance of G protein-coupled receptors (GPCR) have made these receptors excellent drug targets. It is becoming clear that the regulation of the GPCR itself – its location, stability, and signal duration – is a key component of the signaling process [ 1 , 2 ] The length of a GPCR signal can be modulated by receptor desensitization (decrease in receptor responsiveness) and receptor internalization (trafficking of receptors to endocytotic vesicles). The cell can also respond to prolonged activation by upregulating compensatory pathways. For example, prolonged signaling through a G i -coupled receptor inhibits adenylyl cyclase (AC), while paradoxically increasing the ability of the G s -coupled pathway to stimulate AC, a phenomenon known as AC superactivation [ 3 ]. Such regulatory mechanisms may contribute to the development of drug tolerance and dependence, including the response to chronic opiate use [ 4 ]. The complex effects of drugs at multiple receptor subtypes in multiple tissues have made it difficult to isolate the relative contributions of GPCR regulation, ligand binding, effector coupling, drug metabolism, and cellular downregulation machinery. Even if two receptors couple to the same signaling pathway, the physiological effects of their activation could vary tremendously depending on the pharmacokinetics of the ligands, the cell type expressing the receptors, and the interactions with desensitization mechanisms. An engineered family of receptors that share the same ligand binding and effector coupling, yet have discrete mutations that cause them to internalize or desensitize differentially, would help pinpoint the physiological consequences of GPCR desensitization. This is especially important in the light of recent evidence showing dramatically different endocytosis and signaling profiles of m u o pioid r eceptors (MOR) in response to different ligands [ 5 , 6 ]. In addition, understanding the signals that allow transmembrane proteins to be more or less resistant to endocytosis will improve our understanding of endocytosis as a general regulatory mechanism, as it has been implicated in the regulation of signaling of growth factor receptors [ 7 , 8 ] and ion channels [ 9 - 11 ]. Our laboratory has engineered a G i -coupled receptor that is insensitive to endogenous ligands but can still respond to the synthetic small-molecule agonist spiradoline [ 12 , 13 ]. This r eceptor a ctivated s olely by a s ynthetic l igand (RASSL) was based on the kappa opioid receptor (KOR). In the original RASSLs, exchanging the second extracellular loop of the KOR with the corresponding sequence from the delta opioid receptor, and making an additional point mutation (Q297E), resulted in a receptor with 1/2,000 of the response to dynorphin and other endogenous peptides relative to the wild-type KOR. However, the response of this RASSL to spiradoline was not altered. RASSLs can be expressed in a tissue-specific manner in transgenic mice, allowing direct control of G i -mediated physiological responses such as heart rate [ 14 ]. It has also been recently used to help identify the mammalian sweet receptor by expressing it in mouse taste buds [ 15 ]. To investigate the endocytosis and desensitization of GPCRs, we have since developed four new RASSLs. To visualize these RASSLs in living cells, we fused the green fluorescent protein (GFP) to the N-terminus (outer portion) of the RASSL resulting in Rog ( R ASSL o pioid g reen). Given the well-documented role of the C-terminal region in the desensitization and internalization of GPCRs [ 1 , 16 - 18 ], we made a series of C-terminal mutant RASSLs designed to desensitize and internalize at different rates. This novel receptor system offers an opportunity to test specific hypotheses about the relationship between receptor sequence and receptor regulation, without requiring the use of multiple ligands that might have different effects on the signal and the regulation of receptors. Because RASSLs lack endogenous agonists, they allow greater control of the timing and specificity of activation than is possible with endogenous receptors. In these studies, we test how the removal of phosphorylation sites from the C-terminal regions of a G i -coupled RASSL alters the receptor's internalization, desensitization, and induction of AC superactivation. Since it is well established that the endogenous mu and kappa opioid receptors differ in these properties, we also explore the regulation of kappa opioid RASSLs bearing specific portions of the mu opioid receptor C-terminal sequence. The cell culture experiments presented here provide a basis for in vivo studies in complex tissues such as the nervous system. Insight gained through these experiments may help explain the differences seen in vivo between different endogenous G i -coupled receptors, improving our understanding of the contribution of receptor regulation to the physiological response to agonists and our overall understanding of GPCR signal regulation. Results Rog, a GFP-tagged RASSL, signals appropriately Although an N-terminal GFP tag does not interfere with the function of the human KOR [ 19 ], we wanted to confirm that this tag does not modify the signaling properties of Rog, a KOR-based RASSL. Rog was transiently transfected into CHO cells along with a chimeric G qi5 protein [ 20 ] that couples to G i -coupled receptors but signals through the G q pathway and therefore stimulates calcium release. Using this transient calcium response as a measure of G i activation, we showed by FLIPR assay that Rog responded dose-dependently to spiradoline, but not to a range of doses of dynorphin, the endogenous ligand that activates the wild-type KOR (Figure 2A ). Therefore, Rog, like its predecessors Ro1 and Ro2 [ 12 ], meets the criteria for a RASSL. We evaluated all of the engineered receptors for their ability to affect cAMP formation. G i signaling decreases intracellular cAMP levels by directly inhibiting adenylyl cyclase. Activation of these G i -coupled receptors with spiradoline was therefore expected to inhibit forskolin-induced cAMP accumulation in a dose-dependent manner. Indeed, under basal conditions, 15-min treatment with spiradoline activated Rog, Rog-A, Rog-μ and Rog-μA, as indicated by inhibition of forskolin-induced accumulation of cAMP (Figure 5 , Table 1 ). Despite differences in C-terminal amino acid sequence, the RASSLs showed no significant differences in their ability to inhibit cAMP accumulation after acute activation with spiradoline (Table 1 ). EC50 and cAMP inhibition values for all of the RASSLs were similar to those seen with the human KOR in the same assay. In a representative experiment, the EC50 for KOR was 0.91 nM spiradoline and cAMP inhibition was 68.6%. Rog is internalized by agonist treatment The GFP tag on Rog allows direct observation of agonist-induced receptor internalization by confocal microscopy (Figure 2B ). In untreated cells, the receptor was visible primarily on the plasma membrane. One hour after spiradoline treatment (10–100 μM), the receptor was observed in bright, punctate intracellular vesicles. Dynorphin (100 μM), in contrast, did not lead to significant internalization of the receptor (Figure 2B ). An ELISA that detects only cell-surface receptors was used to quantify the extent of receptor internalization after spiradoline treatment. With increasing doses of spiradoline, fewer receptors were detected on the cell surface, culminating in an approximately 45% loss of cell-surface receptors at the maximal dose of 100 μM (Figure 2C ). The same dose of spiradoline resulted in a similar 47% loss of KOR from the cell surface (not shown). The time course of internalization in response to 1 μM spiradoline was relatively rapid, with significant receptor loss apparent within 5 min (Figure 2C ). Maximal receptor loss was detected approximately 20 min after agonist treatment began. Rog-A is resistant to agonist-induced internalization To determine the role of C-terminal phosphorylation sites in receptor regulation, we examined spiradoline-induced internalization of Rog-A, a mutated version of Rog in which four C-terminal phosphorylation sites were mutated to alanine (Figure 1 ). HEK293 cells stably transfected with Rog-A were treated with 10 μM spiradoline, a dose sufficient to cause internalization of most Rog receptors (Figure 3A , left). One hour after spiradoline treatment, most Rog-A receptors appeared to remain in the membrane (Figure 3A , center). Quantification by cell-surface ELISA showed significantly less loss of cell-surface receptors for Rog-A than for Rog at spiradoline doses of 0.1–100 μM (Figure 3B ). ANOVA indicated a main effect of drug dose (F 10,20 = 66.53, p < 0.0001) and a main effect of receptor type (F 1,20 = 55.29, p < 0.0001). As observed with Rog, maximal internalization of Rog-A in response to 1 μM spiradoline occurred after 20 min of drug treatment (Figure 3B ). However, in contrast to Rog, fewer than 10% of the Rog-A receptors were internalized at that time point. ANOVA of the time course data indicated a main effect of length of treatment (F 11,47 = 11.39, p < 0.0001), a main effect of receptor type (F 1,47 = 203.16, p < 0.0001), and an interaction between receptor type and treatment length (F 11,47 = 2.27, p < 0.02). These results suggest that C-terminal phosphorylation promotes receptor internalization. Activation of Rog-A inhibits cAMP as fully as Rog (Table 1 ), indicating that the reduced internalization does not alter acute signaling. Rog-μ more readily internalizes in response to spiradoline Since the mu opioid receptor (MOR) internalizes more readily than the KOR, we made Rog-μ, a chimeric receptor in which the entire intracellular portion of the C-terminus was replaced with the corresponding MOR sequence (Figure 1 ). Rog-μ was expected to internalize to a greater extent than Rog in response to spiradoline. Confocal microscopy showed nearly complete internalization of Rog-μ after one hour of treatment with 10 μM spiradoline (Figure 3A , right). A cell-surface ELISA revealed 25–30% internalization of Rog-μ at low doses of spiradoline, ranging from 0.01 to 0.1 μM (Figure 3C ). Little internalization of Rog or Rog-A has been observed at these doses (Figures 3B and 3C ). At higher doses of spiradoline, no difference in internalization between Rog and Rog-μ was observed. ANOVA indicated a main effect of drug dose (F 9,52 = 55.79, p < 0.0001) and an interaction between receptor type and drug dose (F 9,52 = 3.89, p < 0.0008). Post-hoc Scheffé analysis shows significant differences between Rog and Rog-μ at 0.01 μM (p = .024) and 0.1 μM (p = .016) doses of spiradoline. When cells were treated with 1 μM spiradoline for differing lengths of time, there was no detectable difference in the time course of internalization of Rog and Rog-μ (Figure 3C ). ANOVA indicated a main effect of time (F 11,48 = 53.20, p < 0.0001), with no effect of receptor type. There was also no difference in cAMP inhibition after spiradoline activation of Rog and Rog-μ (Table 1 ). Constitutive internalization of Rog-μA A variant of Rog-μ, known as Rog-μA, also has MOR sequence at the C-terminus, but five serine and glutamic acid residues at the C-terminus were mutated (Figure 1 ). These mutations were predicted to render Rog-μA more resistant to internalization than Rog-μ [ 18 ]. However, in several independent stably and transiently transfected cell lines (HEK293 and rat1a), Rog-μA always had significantly lower cell-surface expression than other receptors. As shown by cell-surface ELISA of one representative group of stably transfected HEK293 lines, Rog-μA was expressed at 28% of the level of Rog, and 41% of Rog-μ (Figure 4A ). Despite the low cell-surface expression, Rog-μA signals as well as the other RASSLs after acute spiradoline treatment (Table 1 ). Since cell-surface expression of a GPCR can be stabilized by the addition of antagonist [ 21 , 22 ], we examined the effect of the KOR antagonist norBNI on cell-surface expression of Rog-μA. Antagonist treatment nearly doubled the amount of Rog-μA detected in the membrane (Figure 4A ; p < 0.005; F 1,6 = 27.00). It also increased the cell-surface expression of Rog-μ, but to a lesser degree (p < 0.05, F 1,6 = 11.54). In contrast, it had no effect on the cell-surface expression of Rog. The increase in membrane expression of Rog-μA after antagonist treatment was confirmed by confocal microscopy. Under basal conditions, little Rog-μA was seen in the plasma membrane, although the receptor was readily detected in other areas of the cell (Figure 4B ). After overnight treatment with 10 μM norBNI, most of the Rog-μA was seen in the plasma membrane (Figure 4B ). In contrast, untreated Rog receptors were primarily located in the plasma membrane (Figures 2B , 4B ), and norBNI treatment had little effect on their localization (Figure 4B ). These experiments suggest that Rog-μA may be constitutively downregulated and rapidly cycled in and out of the plasma membrane. Rog-A is more resistant to desensitization In addition to regulation by internalization, a GPCR signal can be modulated by desensitization: uncoupling from the signaling effectors after continuous agonist stimulation. To explore desensitization directly, we briefly pretreated each RASSL with 1 nM spiradoline for 15 min, and examined inhibition of cAMP accumulation in response to a variety of doses of spiradoline. The low pretreatment dose had caused no receptor internalization detectable by ELISA-based assays. Pretreatment reduced the responsiveness of Rog receptors to spiradoline (Figure 5A , Table 1 ). The same maximal inhibition of cAMP accumulation was observed, but the dose response curve was shifted approximately 10-fold, with the EC50 for Rog shifting from 0.41 nM to 4.32 nM spiradoline after spiradoline pretreatment. Pretreatment of Rog-A with the same dose of spiradoline, however, did not significantly affect the response of the cells to subsequent treatment (Figure 5A , Table 1 ). The EC50 for spiradoline after 1 nM spiradoline pretreatment of Rog-A was 0.42 nM, compared to 0.41 nM for vehicle-treated cells. Spiradoline pretreatment shifted the EC50 of Rog-μ to 2.05 nM (Figure 5A , Table 1 ). Notably, the maximal response of pretreated Rog-μ-expressing cells was less than half that of untreated cells, indicating a decreased efficacy of Rog-μ signaling through the G i pathway. Spiradoline pretreatment strongly reduced the response of Rog-μA to further spiradoline treatment (Figure 5A , Table 1 ). In fact, it appears that the response to spiradoline in pretreated Rog-μA cells is so low that the dose range tested (up to 100 nM) does not yield a maximal inhibition of cAMP, and no sigmoidal dose-response curve can be fitted to these data. Therefore, we cannot calculate an accurate EC50 for desensitized Rog-μA receptors. However, assuming that the maximal response occurs at doses higher than 100 nM spiradoline, we can estimate that the EC50 would be at least 13.44 nM. This indicates that Rog-μ and Rog-μA receptors desensitize readily. For these receptors, the dose of spiradoline required to achieve the EC50 is significantly lower than the dose required to internalize the cell-surface receptors (Figures 4 , 5 ). While only a fraction of the total surface receptor pool needs to be activated to activate G i maximally, a much larger fraction of the receptor population must be internalized before it can be accurately measured. AC superactivation is independent of receptor internalization Chronic treatment of cells expressing G i -coupled receptors with agonist results in a compensatory increase in the activity of AC and, therefore, an increased accumulation of cAMP in response to the same dose of forskolin [ 3 ]. We examined the development of this AC superactivation in cell lines transiently expressing the RASSL variants. Forskolin (10 μM) stimulates twice as much cAMP in Rog-expressing cells treated with 10 nM spiradoline for 18 hours, compared to cells acutely treated with forskolin alone (Figure 5B ; p < 0.005, F 1,10 = 17.72). A similar degree of superactivation was seen in cells transfected with the wild-type KOR, indicating the same cellular response to prolonged G i signaling through both Rog and KOR. Overnight treatment of Rog-A-expressing cells with spiradoline, followed by stimulation with 10 μM forskolin, resulted in a slightly smaller increase in cAMP (Figure 5B ; p < 0.05, F 1,10 = 9.52). Notably, cells expressing Rog-μ and Rog-μA receptors showed no evidence of AC superactivation after 18 h of spiradoline pretreatment. These data show that receptors that desensitize and internalize more readily at the receptor level, such as Rog-μ and Rog-μA, do not induce compensations in an opposing signaling pathway. Although little internalization of these RASSLs has been observed at these low doses of spiradoline, we wanted to ensure that the AC superactivation data could not be explained by differences in receptor internalization. Therefore, we performed an analysis of cell-surface receptor expression in parallel with the cAMP response experiment. Cells were plated and treated with 10 nM spiradoline for 18 hours exactly as described above. Then the cells were fixed and a cell-surface ELISA was performed. In general, 7–10% of the receptors were internalized by this treatment, but there were no significant differences between receptor types (Figure 5C ). ANOVA showed a significant treatment effect (F 1,18 = 8.22, p < 0.01), but no effect of receptor type (p > 0.99). Discussion We have engineered a series of RASSLs that inhibit cAMP after acute activation by spiradoline with equal efficacy but differ dramatically in cellular location and responses to chronic drug treatment. Mutation of phosphorylation sites on the C-terminus to alanines resulted in a receptor that was relatively resistant to internalization in the presence of moderate doses of agonist (Rog-A, Figure 3 ) and showed no significant desensitization after pretreatment with a low dose of agonist (Figure 5A ). This is consistent with recent findings that mutation of a single serine to alanine is sufficient to block internalization and desensitization of the KOR, since this mutation removes a residue that is required for G protein receptor kinase (GRK2) phosphorylation [ 23 ]. While these studies highlight the importance of GRK phosphorylation of GPCRs in mediating receptor internalization and desensitization, it is notable that partial internalization of Rog-A was still detected in response to higher doses of spiradoline, indicating that the receptor can be internalized through different mechanisms. Reports of GPCR endocytosis in the absence of GRK phosphorylation [ 24 - 26 ] suggest that the removal of C-terminal phosphorylation sites may reduce the affinity of the receptor for proteins that mediate endocytosis without preventing the protein-protein interactions that are essential for internalization. Rog-μ, the MOR/KOR chimeric mutant, was more sensitive to agonist-induced desensitization (Figure 5A ) and internalization (Figure 3 ) than Rog. This is consistent with observations that the MOR internalizes and desensitizes more readily than the KOR. Surprisingly, mutation of C-terminal phosphorylation sites on the MOR/KOR chimera to form Rog-μA did not inhibit desensitization (Figure 5A ). Rog-μA has low basal surface expression, although the GFP-tagged receptor can be seen throughout the cell (Figure 4B ). It is unlikely that the extracellular mutations in Rog-μA are responsible for the unusual internalization pattern of this receptor. The extracellular mutations in Rog-μA are identical to the ones in Rog, which was shown to reach the cell surface and respond to agonist the same as GFP-tagged wild-type KOR (Figure 2 ). The intracellular pool of Rog-μA is not due to receptor misfolding or abnormal sorting, because some receptor was detected on the membrane (Figure 4A ) and the receptor showed normal agonist-induced signaling (Figure 5A , Table 1 ). The observation that the addition of an antagonist can "rescue" the low cell-surface expression of Rog-μA (Figure 4A ) further suggests that misfolding is not responsible for the decrease in cell surface expression. There are several potential mechanisms for the increase in cell-surface expression after antagonist treatment. One possibility is that the antagonist, norBNI, acts as a molecular chaperone, entering the cell, binding to the receptor in intracellular compartments, and bringing it to the membrane. Ligands can act as pharmacological chaperones for the delta opioid receptor, facilitating receptor maturation and export from the endoplasmic reticulum [ 21 ]. However, there are no reports that the norBNI antagonist is cell-permeable. Another possibility is that norBNI acts as an inverse agonist, stabilizing cell-surface receptors in an "off" conformation, making them inaccessible to GRKs and arrestins, which usually interact only with active receptors. This would suggest that in the absence of norBNI, Rog-μA may be constitutively active. However, the receptor still signals robustly in response to spiradoline (Figure 5A ), so it cannot be fully active in the absence of ligand. It is also possible that, under basal conditions, Rog-μA has a higher than normal affinity for GRK or arrestin, but not the G proteins. This would result in constitutive turnover – the receptor constantly cycling in and out of the membrane – in the absence of constitutive signaling. The idea that this receptor is especially sensitive to the desensitization and internalization machinery is borne out by the observation of extensive desensitization in response to pretreatment with a low dose of agonist (Figure 5A ). It will be interesting to investigate the physiological consequences of this apparent constitutive internalization and rapid desensitization in animal models. Although most of the C-terminal sequence of Rog-μA is derived from the MOR, the MOR does not exhibit either constitutive turnover or abnormally low membrane expression. There is some evidence for partial basal internalization of the similar Rog-μ receptor (Figure 4A ), although Rog-μ appears to be expressed predominantly at the cell surface under basal conditions. Since Rog-μ and Rog-μA differ at only five amino acids, some of those five residues must be responsible for the increased turnover of Rog-μA. Although phosphorylation of T394 has been reported to be required for desensitization of the MOR [ 18 ], subsequent reports have shown that mutating T394 to alanine facilitates the internalization and resensitization of the receptor [ 27 ]. This suggests that phosphorylation of T394 may be a membrane retention signal and that the T394A mutation in Rog-μA is responsible for the constitutive endocytosis observed in this study. Mutagenesis of individual amino acids in this region may allow the identification of the specific residue(s) responsible for the constitutive internalization of Rog-μA. The differences in AC superactivation between our RASSLs indicate another layer of complexity in GPCR signaling. Previous studies suggest an inverse correlation between the ability of an opioid receptor to undergo ligand-activated endocytosis and its ability to induce AC superactivation by chronic signaling [ 28 ]. The induction of superactivation by Rog is consistent with this idea. Rog-μ, which internalizes and desensitizes readily, failed to induce any AC superactivation after chronic activation (Figure 5B ). Similarly, Rog-μA, which may undergo constitutive endocytosis, did not induce AC superactivation after chronic administration of spiradoline. However, Rog-A, which was predicted to have enhanced superactivation due to its resistance to endocytosis, had levels of superactivation comparable to those seen with Rog. It is possible that Rog induces maximal superactivation, and the cell cannot respond to Rog-A signaling with any additional superactivation. Our results indicate that internalization does not directly induce AC superactivation. The dose of spiradoline (10 nM over 18 hours) used to induce strong AC superactivation in these experiments causes only minimal internalization of all of the receptors (Figure 5C ). Moreover, there is no significant difference in degree of internalization among the different receptor types, although they show profound differences in superactivation. This suggests that the cellular mechanisms underlying AC superactivation and receptor internalization are independent. AC superactivation has been attributed to upregulation of AC proteins induced by Gβγ protein subunits interacting directly with GRK2/3 proteins [ 29 , 30 ]. Therefore, the same mutations that prolong Rog-A signaling and inhibit endocytosis may also prevent the receptor from interacting with GRK2/3 proteins and subsequently activating Gβγ-mediated signaling events. Alterations in desensitization characteristics are unlikely to alter AC superactivation because of the drastic differences in time course underlying these distinct phenomena. Receptor desensitization happens on a scale of minutes, while AC superactivation is the result of much longer term chronic receptor activation. Therefore, a decrease in G i signaling due to a more desensitized receptor is unlikely to have a significant effect on AC superactivation over the much longer time course used in these experiments. The additional possibility exists that altering C-terminal residues on the RASSL could increase the ability of receptors to couple to G o , resulting in perceived changes in AC superactivation [ 31 ]. However, if this were the case, one would expect to see a shift in the dose response curve for cAMP inhibition between Rog, Rog-A, Rog-μ and Rog-μA that is not observed in any of our experiments. Further studies with this engineered receptor system in vivo may clarify the complex relationship between ligand dependent endocytosis, interaction of a GPCR C-terminus with GRK2/3, desensitization, and superactivation of AC. The ability of these RASSLs to induce different degrees of AC superactivation may have important physiological consequences in vivo . Interestingly, when a RASSL with a C-terminus corresponding to the wild-type human KOR was expressed at high levels in the hearts of transgenic mice, the mice developed a lethal cardiomyopathy [ 32 ]. One possible explanation is that basal signaling of the RASSL in mouse heart may increase G s signaling through AC superactivation. G s signaling has long been associated with heart failure, so AC superactivation may be responsible for the cardiomyopathy. Rog-μ and Rog-μA, RASSLs that do not induce superactivation, could be used to test this hypothesis and to study the consequences of AC superactivation in other tissues. Internalized opioid receptors can be either degraded or recycled back to the membrane. The receptors that return to the membrane are stripped of arrestin, phosphates, and ligand, and are resensitized to ligand. Recent evidence demonstrates that the C-terminus is crucial for directing internalized receptors either into the degradative lysosomal pathway or back to the plasma membrane [ 27 , 33 ]. We expect that several of our engineered RASSLs should also differ in their post-endocytotic fate. It is likely that the complex mechanisms governing GPCR endocytosis, recycling, desensitization and AC superactivation will be regulated differently in different cell types. The RASSLs described here exhibit similar properties in several different mammalian cell lines we tested (rat1a, CHO and HEK293), but their properties may change in specific cell types or under specific physiological conditions. One potentially fruitful avenue for future investigations would be to target different RASSLs to particular cell types in vivo . This would allow a thorough investigation of the interplay between receptor sequence and cell-type specific mechanisms of receptor regulation. The development of a toolbox of engineered RASSLs that differ in internalization and desensitization raises several possibilities for future research and clinical investigations. Growing evidence points to a link between receptor dynamics and the potential for drugs to elicit tolerance or dependence, especially for opioid receptors [ 28 , 34 ]. Here, we present a system that allows the same drug to activate different receptors that have small, well-defined variations in sequence. The efficacy and potency of spiradoline is similar for Rog, Rog-A, Rog-μ and Rog-μA (Table 1 ); only the desensitization and internalization responses vary. Specifically, agonist-induced internalization is reduced in Rog-A and enhanced in Rog-μ, while Rog-μA shows agonist-independent internalization. Rog-A shows no desensitization after a brief spiradoline pretreatment, while the same treatment reduces the potency of spiradoline at Rog-μA and reduces efficacy at Rog-μ. Longer spiradoline pretreatment induces normal AC superactivation at Rog-A, but does not affect the AC response in Rog-μ or Rog-μA cells. A family of engineered GPCRs that do not respond to endogenous ligands has enormous potential for selectively controlling G protein signaling in specific tissues without interfering with endogenous processes. Rog-A, a long-signaling RASSL, has several interesting implications for in vivo signal engineering. The basic RASSL, Ro2, shows rapid and extensive physiological desensitization [ 14 ], making it difficult to use in any therapeutic context where repeated activation of the receptor would be necessary. Rog-A, with its reduced desensitization, could allow continued physiological efficacy of repeated drug treatments. Comparison of the regulation and signaling of Rog, Rog-A, Rog-μ, Rog-μA, and future variants will contribute to the growing understanding of how GPCR signals are dynamically modulated. Study of these RASSLs in vivo will help solidify the elusive links between the receptor amino acid sequence, cell biology, and complex physiology. Methods Construction of mutant receptors All receptors were based on the human kappa-opioid RASSL called Ro2 [ 12 ]. The GFP-tagged version of the RASSL has been named "Rog" for R ASSL o pioid with G FP tag. Rog was made by inserting the coding sequence for emerald GFP (Packard) at the N-terminus of the receptor, after a FLAG tag (DYKDDDDV) and the first eight amino acids of the RASSL. To create Rog-A, two serines and two threonines in the C-terminal region of the receptor were mutated to alanines (Figure 1 ). In the human kappa opioid receptor (KOR), the mutated residues correspond to S356A, T357A, S358A and T363A. The exact location of those residues in our RASSL construct, and the complete sequence of all RASSL variants can be found on our web site . For both Rog-μ and Rog-μA, the final 35 amino acids (345–380) of Rog were replaced by 47 C-terminal residues from the rat mu opioid receptor (MOR). Rog-μA contains the following additional modifications to the rat MOR C-terminus: T383A, E388Q, E391Q, E393Q and T394A. For each receptor, a schematic design and a C-terminal amino acid sequence alignment is shown in Figure 1 . All constructs were sequenced to verify the mutations. Expression of RASSLs in mammalian cells HEK293 cells were grown in culture to 60–80% confluence and then transfected using Lipofectamine Plus (Invitrogen, Carlsbad, CA). The RASSL construct contained a cytomegalovirus promoter to drive mammalian expression, and a neomycin-resistance gene to allow selection of stable cell lines. Experiments on transiently transfected cells were performed approximately 48 h after transfection. To create stable cell lines, transfected cells were selected with G418 (500 μg/ml, Invitrogen) for 10–14 days. Individual colonies showing green fluorescence were selected and grown under maintenance doses of G418 (250 μg/ml). Receptor expression was confirmed visually by fluorescence microscopy, and by an enzyme-linked immunoadsorbent assay (ELISA, see below). Cell-surface ELISA Cell-surface expression of receptors was confirmed by an ELISA that detects only extracellular FLAG tag, which labels the N terminus of all RASSLs. This assay therefore quantifies only receptors that are in the membrane at the time of labeling, without providing detailed localization data about those receptors. Cells were plated at 100,000 cells/well on to 24-well plates coated with poly-d-lysine. Cultured cells were fixed in 4% paraformaldehyde for 10 min at 4°C, washed in phosphate-buffered saline (PBS), and then incubated in 1 μg/ml M1 anti-FLAG antibody (Sigma, St. Louis, MO) for 1 h at room temperature. They were washed again in PBS with 1 mM CaCl 2 and incubated for 30 min at room temperature in secondary antibody (1:1000 goat anti-mouse conjugated with horseradish peroxidase, Biorad, Chicago, IL), then washed three times in PBS plus CaCl 2 . To develop the reaction, 0.25 ml of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) liquid substrate (Sigma) was added to each well. After 15–60 min, 200 μl from each well was transferred to a 96-well plate and the optical density was read at 410 nm. To quantify agonist-induced internalization, cells were treated with various doses of spiradoline in medium for 10–120 min. The medium was removed and the cells were fixed and processed as described above. Vehicle treated cells on each plate were used to calculate a "maximum" cell-surface expression for that plate. All other treatment conditions on that plate were then normalized to this maximum to determine "percent internalization." Each experiment included 3–6 replicates per condition and was repeated at least 3 times. After values for cell-surface expression of each receptor were calculated and normalized, receptor expression was compared using two-way ANOVA (StatView v. 5.0, SAS Institute, Cary, NC). For dose response studies, receptor and dose were independent factors. For time course studies, receptor and length of treatment were independent factors. cAMP accumulation assay The degree of cAMP inhibition in spiradoline-treated HEK-293 cells transiently expressing RASSL variants was measured with the CatchPoint cAMP ELISA kit (Molecular Devices, Sunnyvale, CA). Cells were plated at 5 × 10 4 /well into 96-well plates coated with poly-d-lysine. The next day, cells were rinsed in Krebs-Ringer bicarbonate buffer with glucose (KRBG, Sigma). Cells were then incubated in pre-stimulation buffer containing a phosphodiesterase inhibitor (0.75 mM 3-isobutyl-1-methylxanthine in KRBG buffer) for 10 min at room temperature to inhibit cAMP degradation. cAMP production was stimulated by the addition of 50 μM forskolin to all cells. At the same time, various doses of spiradoline in PBS were added to cells to establish a dose-response curve. After a 15-min drug treatment at 37°C, the cells were lysed and cAMP accumulation was assayed according to the CatchPoint protocol. Inhibition of cAMP by spiradoline was determined by comparison to cells treated with forskolin alone. For each experiment, 3–6 wells per condition were averaged, and EC50 and percent inhibition values for each receptor were determined by fitting curves for each independent experiment using SOFTmax PRO v. 4.0.1 (Molecular Devices). Data for 3–8 independent experiments were averaged to determine the EC50 and maximum cAMP inhibition for each receptor and condition. For desensitization assays, a pretreatment dose (1 nM) of spiradoline diluted in sterile PBS was added to the cells, which were then incubated for 10 min at 37°C. The cells were rinsed 4 times in KRBG and then stimulated and assayed for cAMP as described above. AC superactivation was determined by measuring forskolin-stimulated (10 μM) cAMP after treatment with either 10 nM spiradoline or vehicle for 18 h. AC superactivation data were expressed as a percent increase in forskolin-stimulated cAMP relative to vehicle-pretreated cells expressing the same receptor. Conditions and receptors were compared using a two-way ANOVA with receptor and treatment condition as independent factors. Confocal microscopy HEK293 cells stably expressing receptor constructs were plated at a density of 500,000 cells/ml onto glass Labtek II chamber slides (Fisher Scientific, Pittsburgh, PA) coated with poly-d-lysine. The following day, the cells were treated with agonist (spiradoline or dynorphin A 1–13) for typically one hour, or antagonist (NorBNI) overnight and briefly washed in PBS. The PBS was removed and replaced with 1 ml of cold 4% paraformaldehyde in PBS. The cells were fixed at room temperature for 10 min, washed with PBS, and then mounted in Vectashield (Vector Laboratories, Burlingame, CA) under cover slips. For confocal imaging on a Bio-Rad MRC 600 microscope, typical images were taken with a 40–60× oil immersion objective lens, subject to 5× Kalman filtering. The microscope operator was blind to both cell line and treatment condition. Fluorometric imaging plate reader (FLIPR) assay All receptors tested were transiently transfected using Lipofectamine into CHO cells in conjunction with the chimeric G protein G qi5 [ 20 ] in a 5:1 molar ratio of DNA. G qi5 is a chimeric G protein alpha subunit that has the G αq wild-type sequence except for the C-terminal 5 residues, which were changed to the corresponding G αi sequence. This allows G i -coupled receptors to signal through the G q pathway, resulting in a signal that can be detected using calcium-sensitive reagents. The following day, the cells were plated in a 96-well plate (50,000 cells per well) and allowed to grow for 24 h before being incubated with the calcium-sensitive dye fura-3 for 1 h. The assay was performed as described [ 12 ] using a range of dilutions of either spiradoline or dynorphin A 1–13 peptide (Sigma). All experiments were performed in triplicate. Authors' contributions KSL and MDL created the constructs and cell lines used here, designed and conducted the experiments; analyzed the data; and drafted the manuscript. HHE maintained cell lines and participated in making constructs, internalization assays and confocal microscopy. BRC conceived of the study and participated in its design and interpretation. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551613.xml |
551612 | Comparison of artificial neural network and logistic regression models for prediction of mortality in head trauma based on initial clinical data | Background In recent years, outcome prediction models using artificial neural network and multivariable logistic regression analysis have been developed in many areas of health care research. Both these methods have advantages and disadvantages. In this study we have compared the performance of artificial neural network and multivariable logistic regression models, in prediction of outcomes in head trauma and studied the reproducibility of the findings. Methods 1000 Logistic regression and ANN models based on initial clinical data related to the GCS, tracheal intubation status, age, systolic blood pressure, respiratory rate, pulse rate, injury severity score and the outcome of 1271 mainly head injured patients were compared in this study. For each of one thousand pairs of ANN and logistic models, the area under the receiver operating characteristic (ROC) curves, Hosmer-Lemeshow (HL) statistics and accuracy rate were calculated and compared using paired T-tests. Results ANN significantly outperformed logistic models in both fields of discrimination and calibration but under performed in accuracy. In 77.8% of cases the area under the ROC curves and in 56.4% of cases the HL statistics for the neural network model were superior to that for the logistic model. In 68% of cases the accuracy of the logistic model was superior to the neural network model. Conclusions ANN significantly outperformed the logistic models in both fields of discrimination and calibration but lagged behind in accuracy. This study clearly showed that any single comparison between these two models might not reliably represent the true end results. External validation of the designed models, using larger databases with different rates of outcomes is necessary to get an accurate measure of performance outside the development population. | Background In recent years, outcome prediction studies have become the avante garde in many areas of health care research, especially in critical care and trauma. However acceptable models for outcome prediction have been difficult to develop [ 1 ]. According to Wyatt and Altman, to be useful, a predictive model must be simple to calculate, have an apparent structure and be tested in independent data sets with evidence of generality [ 2 ]. While this is a high standard, availability and popularity of portable computers, deprioritize the need for simplicity of the model and having an apparent structure. Artificial neural networks (ANNs) are mathematical constructs modeled on interconnection of nodes (neurons) giving a loose association with the animal nervous system. [ 3 ] ANNs employ nonlinear mathematical models to mimic the human brain's own problem-solving process. Just as humans apply knowledge gained from past experience to new problems or situations, a neural network takes previously solved examples to build a system of "neurons" that makes new decisions, classifications, and forecasts. [ 4 ] ANNs are complex and flexible nonlinear systems with properties not found in other modeling systems. These properties include robust performance in dealing with noisy or incomplete input patterns, high fault tolerance, and the ability to generalize from the input data [ 5 ]. Neural networks excel at applications where pattern recognition is important, and precise computational answers are not required, such as forecasting weather, stock predicting, or speech recognition [ 6 ]. Reports in medical literature suggest that ANN models are applicable in diagnosing diseases such as myocardial infarction [ 7 , 8 ] pulmonary emboli detection [ 9 ], gastrointestinal hemorrhage [ 10 ], waveform analysis of EKGs [ 11 ], EEGs [ 12 , 13 ], and radiographic images [ 14 ]. ANNs have also been successfully applied in clinical outcome prediction of trauma mortality [ 1 , 15 ], surgical decision making on traumatic brain injury patients [ 16 ], recovery from surgery [ 17 , 18 ], outcome in pediatric meningococcal disease [ 19 ] and transplantation outcome [ 20 ]. Lang EW et al have compared ANN with Logistic Regression in prediction of outcome after severe head injury and concluded that the differences in the results obtained with the two models were negligible [ 21 ]. Almost all of the published articles indicate that the performance of ANN models and logistic regression models have been compared only once in a dataset and the essential issue of internal validity (reproducibility) of the models has not been addressed. The objective of this study was to compare the performance of ANN and multivariate logistic regression models for prediction of mortality in head trauma based on initial clinical data and whether these models are reproducible. We used different variables even if they were interdependent. Methods Study population Among 8452 trauma patients' records admitted to the emergency departments of six major university hospitals in Tehran from 23 August 1999 to 22 September 2000, the records of 1271 patients whose main trauma was head injury, were selected for this study. The selection of head trauma as the main trauma was based on the definition of principal diagnosis in the Uniform Hospital Discharge Data Set (UHDDS). It defines the principal diagnosis as "that condition established after study to be chiefly responsible for occasioning the admission of the patient to the hospital for care". For making determination of the main trauma more practical in the case of ambiguity, hospitalization in the neurosurgical ward was used as an additional guideline. The database was based upon the trauma data registry program began in 1996 in Trauma Research Center, Sina Hospital, a hospital affiliated with the Tehran University of Medical Sciences [ 22 , 23 ]. The study population for this study was comprised of all trauma victims who had been admitted in one of the hospitals for more than 24 hours during the data-gathering period. For dead patients, this time limitation was disregarded, that is, records of all dead patients who were referred to these hospitals were included in the study. We have excluded those transferred to other hospitals or with related missing values. Structured, closed-question data checklists were used for the data gathering process. Three major categories of injury-related information were collected, that is, demographic data, pre-hospital data (if they were available) and in-hospital data. Hospital related data included: vital signs, Glasgow Coma Scale (GCS), Abbreviated Injury Scale (AIS-90 [ 24 ]), clinical findings in accordance with the International Classification of Diseases 10 th revision (ICD-10) as well as the outcome of the patients. Data collection was conducted by a group of trained physicians who had completed special training courses to become familiar with the process of extracting Abbreviated Injury Score (AIS-90) codes and filling out the relevant questionnaires. For quality control (QC) purposes each hospital had a physician, who was responsible for overseeing the data gathering process. In the intubated patients GCS were calculated according to the other portions of the scale by this physician. Finally, a medical practitioner examined all the checklists in order to evaluate and amend them if deemed necessary based on pre-arranged and fixed protocols. Since we were trying to build and compare models for prediction of outcome mainly based on the initial clinical data, only data related to the GCS, tracheal intubation status, age, systolic blood pressure (SBP), respiratory rate(RR), pulse rate(PR), injury severity score (ISS)(upon admission) and outcome were used in our study. In order to prepare the data for the Neural Network software and to enhance the reliability of the data, three variables of systolic blood pressure, respiratory rate and pulse rate were transformed to dichotomous(1,0) variables. Low systolic blood pressure was defined according to the following cutoff points: up to 5 years of age, less than 80 mmHg; and 5 years of age or older, less than 90 mmHg. Respiratory rate of 35 per min and pulse rate of 90 per min were selected as limits for definition of tachypnea and tachycardia. Other variables including GCS, age, systolic blood pressure (SBP) and injury severity score (ISS) variables were also converted from decimal (Base 10) to binary (Base 2). This conversion was carried out in order to render the input data suitable for processing by our ANN software with its default settings. The data and the data format were similar for both ANN and logistic regression models. Development of logistic regression models The dataset was divided randomly into two sets, one set of 839 cases (66% of the whole dataset) for training and 432 cases for testing the model. A model was built using a training set with logistic regression. GCS, tracheal intubation status(dichotomous), age, SBP(dichotomous), RR(dichotomous), PR(dichotomous) and ISS were the independent variables and the outcome (death/survival) was the dependent variable. The logistic regression analyses were performed using Intercooled STATA for windows, Version 6 (STATA Corp., College Station, TX) "logistic" default options. The built logistic model was tested using the testing dataset (432 cases). These steps (randomized division of dataset and regression analysis considering the same variables) were repeated 1000 times. This resulted in 1000 pairs of training and testing datasets (2/3 and 1/3 of the original dataset, respectively) which were saved for further processing by the neural networking. Development of ANN models The ANN used in this study was a standard feed-forward, back-propagation neural network with three layers: an input layer, a hidden layer and an output layer. The input layer consisted of 23 input neurons, the hidden layer consisted of fifteen hidden neurons, and the output layer consisted of one output neuron (Fig. 1 ). The learning rate and momentum for network training were set respectively to 0.25 and 0.9 and the models were run until a minimum average squared error < 0.063 was obtained. The number of the network layers, hidden neurons and the stopping criteria were determined through trial-and-errors process because no commonly accepted theory exists for predetermining the optimal number of neurons in the hidden layer [ 25 ]. Figure 1 Diagrammatic representation of Artifical Neural Network ANN) structure with 23 input nodes in the input layer, 15 nodes in the hidden layer and one node in the output layer. The training and testing datasets were the same as those were used with regression models, thus there was an ANN and a logistic model for each training and testing dataset. PDP++ version 3.1 was used for the artificial neural network analysis as it has a powerful built-in scripting language and is freely available. This software can be downloaded from . Comparison of model performance Discrimination and calibration (goodness of fit) were both measured. Discrimination refers to the ability of a model to distinguish those who die from those who survive. A perfectly discriminating model would assign a higher probability of death to all cases that died than to any case that survived. The discriminatory power of the models was analyzed using the area under the receiver operating characteristic curves (ROC-A(z)). ROC curves were constructed by plotting true positives (patients who died and whom the model predicted as dying [i.e. sensitivity]) versus the false positive fraction (fraction of the patients who lived and were incorrectly classified as dying [i.e. (1 – specificity)]). A ROC-A(z) value of one corresponds to a test that perfectly separates two populations, whereas a ROC-A(z) value of 0.5 corresponds to a perfectly useless test that performs no better than chance. The relative calibration of the models, that is, how accurately the models predicted over the entire range of severity, was compared using Hosmer-Lemeshow (HL) statistics. The HL statistics is a single summary measure of the calibration and is based on comparing the observed and estimated mortality for patients grouped by estimated mortality. The resulting statistic follows a chi-squared distribution, with degrees of freedom equal to two less than the number of groups (10 in this study). The smaller the HL statistics, the better the fit, with a perfectly calibrated model having a value of zero. A probability cut point of 0.5 was used to classify observations as events or nonevents. The overall accuracy ([true positive+true negative]/total) of the final model was determined by comparing the predicted values with the actual events. For each of one thousand pairs of ANN and logistic models (trained and tested on the same datasets), these indices (area under the ROC curves, HL statistics and accuracy rate) were calculated and compared using paired T-tests (P < 0.05). All the statistical analyses were performed using Intercooled STATA for windows, version 6 STATA Corp., College Station, TX) and its downloadable add-on ado files. Some of the scripts used both for the STATA and PDP++ and the designed neural networks can be downloaded from the site of the Tehran University of Medical Sciences . Further details are available from the corresponding author. Results Table 1 shows clinical characteristics of the dataset. The mean age of the study population was (28.5 ± 19) years. 76% of our patients were male and the overall mortality rate was 7.5%. 7.5% of the patients had GCS < 8. Table 1 General Characteristics of the Dataset GCS 13.5 ± 3 Sex 76% Male ISS 9.5 ± 14 SBP 117 ± 23 RR 19 ± 7 PR 86 ± 17 Intubated 2% Age 28.5 ± 19 Mortality 7.5% GCS = Glasgow Coma Scale; ISS = Injury Severity Score; SBP = Systolic Blood Pressure; RR = Respiratory Rate; PR = Pulse Rate As is seen in Table 2 ANN significantly outperformed logistic models in both senses of discrimination and calibration, although from the standpoint of accuracy (cutoff point 0.5), logistic models were superior to ANN models. In 77.8% of cases the area under the ROC curve and in 56.4% of cases the HL statistics for the neural network model were superior to that for the logistic model. In 68% of cases the accuracy of the logistic model was superior to the neural network model. Table 2 Results of Comparing 1000 pairs of Artificial Neural Network (ANN) and Logistic Regression (LR) models LR(95% Confidence Interval) ANN(95% Confidence Interval) P < Area under ROC curve .9538 (.9527 – .9549) .9646 (.9627 – .9665) 0.0000 H-L Statistics 53.13 (46.04 – 60.23) 41.51 (32.92 – 50.11) 0.015 Accuracy Rate 96.37 (96.32 – 96.42) 95.09 (94.93 – 95.24) 0.0000 The confidence intervals of the all the measures show higher variation in the ANN models results. Discussion Prognostic evaluation of the patients without CT scan findings may have limited applicability considering the availability of CT scans in the majority of the trauma centers. Nevertheless, the idea of prognostic models based on initial clinical data at admission is still worth trying and seems to be of practical value in some situations. Although omission of CT data weakens our armamentarium significantly, patients without paraclinical and imaging data make the real trauma scenes at which medical staff should have an evaluation. Actually as neurosurgeons we have initial clinical judgments based on our growing experiences and in many situations they prove to be true. We can expect computers do similar things and help us. In fact, vague situations like what we see in primary evaluation of head trauma patients are where ANN may prove to be superior to traditional linear modeling. This is one of the reasons that although many paraclinical and imaging factors are known to be of significant predictive value in the outcome of the head trauma patients, this study only used clinical measures which are simply available to a physician in the emergency department. Study limitations Calculating ISS and AIS are not simple tasks and needs training. This can reduce the practicability of the results. The pupillary size and reactivity are one of the clinical signs with prognostic value that were not considered in the study due to defect of the main database in this regard. As is seen the mortality rate and the percentage of patients with GCS < 8 are the same (7.5%). This is a coincidence, but emphasizes the need for further studies in the populations with different rates of outcome. The mean GCS was 13.5 ± 3. The standard deviation is over the top score of GCS (15) and means that the GCSs were skewed towards higher levels of consciousness. Not considering the exact time interval between head trauma and admission which is simply available for the medical staff is one of the weak points of this study. The lower intubation rate in this population study related to the distribution of the GCS may show the lower quality of the pre-hospital care prevailing in the hospitals selected for this study. This may render the reproduction of the results using the same network (Downloadable from ) somewhat difficult in other situations where pre-hospital care services are more advanced. Using ISS as an independent variable underlines the role of general trauma in the models used for this study. This was unavoidable, because the original dataset had been a subset of general trauma patients. Comparison of two models Currently, the logistic regression and the artificial neural networks are the most widely used models in biomedicine, as measured by the number of publications indexed in Pubmed as attested by 45646 cases for the logistic regression and 8015 for the neural network. Logistic regression is a commonly accepted statistical tool, which can generate excellent models. Its popularity may be attributed to the interpretability of model parameters and ease of use, although it has limitations. For example, logistic regression models use linear combinations of variables and, therefore, are not adept at modeling grossly nonlinear complex interactions as has been demonstrated in biologic and complex epidemiologic systems. Not withstanding its limitations, neural networks are appealing for a number of reasons, namely; they seem to "learn" without supervision, they can be created by workers with very little mathematical model building experience, and software for building neural networks is now readily available. Neural networks have perhaps a special appeal to the medical community because of their superficial resemblance to the human brain (a structure with which most physicians are comfortable), and seem to promise "prediction" without the difficulties associated with use of mathematics. ANNs are rich and flexible nonlinear systems that show robust performance in dealing with noisy or incomplete data and have the ability to generalize from the input data. They may be better suited than other modeling systems to predict outcomes when the relationships between the variables are complex, multidimensional, and nonlinear as found in complex biological systems. The difficulty in developing models using artificial neural networks is that there are no set methods for constructing the architecture of the network. The most common type of artificial neural networks is the feed-forward back propagation multiperceptron (used in this study). Another limitation of neural network models is that standardized coefficients and odds ratios corresponding to each variable cannot be easily calculated and presented as they are in regression models. Neural network analysis generates weights, which are difficult to interpret as they are affected by the program used to generate them [ 26 ]. This lack of interpretability at the level of individual variables (predictors) is one of the most criticized features in neural network models [ 27 ]. Several early applications of neural networks in medicine reported an excellent fit of the ANN model to a given set of data. The impressive results usually were derived from over fitted models, where too many free parameters were allowed. Linear and logistic regression models have less potential for overfitting primarily because the range of functions they can model is limited. Neural network models require sophisticated software. The complexity and unfamiliarity of ANN has been a major drawback of this technique so far. However, as palmtop computing becomes increasingly powerful and popular, the complexity of ANNs may become less onerous in real-time clinical settings. [ 4 ] Furthermore, there are some theoretical advantages comparing a predictive ANN model over conventional models such as logistic regression. One such advantage is that ANN model allows the inclusion of a large number of variables [ 28 ]. Another advantage of the neural network approach is that there are not many assumptions (such as normality) that need to be verified before the models can be constructed. Although, one of the strengths of ANNs is their ability to still find patterns despite missing data, in this study a dataset with no related missing values was used. Recently the task of comparison between these two models has been addressed from different points of view. Considering the publication bias, several published works in the medical literature have demonstrated the success of the ANN approaches. In a review carried out by Sargent on 28 major studies, ANN outperformed regression in 10 cases (36%), was outperformed by regression in 4 cases (14%) and the 2 methods had similar performance in the remaining cases. Sargent concluded that both methods should continue to be used and explored in a complementary manner. [ 29 ] Gaudart et al. using simulated data, have compared the performance of ANN and linear regression models for epidemiological data and concluded that both had comparable performance and robustness and despite the flexibility of connectionist models (like ANN), their predictions were stable. [ 30 ] This study was primarily designed to compare the performance of an ANN and a multivariable logistic regression analysis with the goal of developing a model for predicting the outcome in head injury and for studying their internal validity (reproducibility). Also setting up a standalone practical model for prediction of mortality in the head trauma patients was a secondary goal of this study. Using freely downloadable software (PDP++) and making the networks and scripts accessible by the researchers can be perceived as an advantage of this study. This study showed that ANN models significantly outperformed logistic models in both senses of discrimination and calibration, although lagged behind in accuracy. It is pointed out that the calibration values should be treated with some caution in this study, since according to Hosmer and Lemeshow, [ 31 ] in describing the statistic, HL statistic should be used where at least one of the predictor variables is continuous. This study clearly shows that in a single comparison of these models based on the same data there is 22.2% chance of getting discrimination results contrary to our findings in majority of comparisons. This ratio is 43.6% for calibration and 32% for the accuracy results. These figures are practically important and imply that any single comparison between these two models cannot reliably represent their final performance. Although considerable efforts, through many trial-and-errors, were made to optimize the design of the network, the designed ANN models could, and should, be further improved. In line with any other predictive models, likewise the findings of this study need to be externally validated. The networks are downloadable and the results can be studied in other study populations with divergent data and different survival ratios. So far, there is no single algorithm that performs better than all other algorithms for any set of given head injury data. To this end, there is room for much more work to be done before a definite conclusion can be reached. Potential clinical use Should the results be reproducible in other populations, using a simple preprogrammed calculator (or other programmable computing devices) and minimal training of the personnel, this model and similar ones may emerge to be of considerable practical value in triage of the patients. At that time a dedicated instead of general purpose ANN software should be designed for this purpose. The authors concur with the conclusion arrived by Tu [ 25 ] that logistic regression remains the clear choice when the primary goal of model development is to examine possible causal relationships among variables. However, it appears that ANNs or some form of hybrid technique incorporating the best features of both logistic regression and neural network models might lead to development of optimum prediction models for head injured patients. Conclusions In conclusion, this study compared models for the prediction of outcome in head injury using trauma data from hospital registries in Tehran, the data was applied to artificial neural network and multivariable logistic regression analysis. The predictive ability of the artificial neural network model was found to be comparable to that of the logistic regression model. Specifically, the ANN models significantly outperformed logistic models in both senses of discrimination and calibration but lagged behind in accuracy. Although the performance of the models were studied when the models were applied to the different samples of the original population study, external validation is necessary to get an accurate measure of performance outside the development population. Studies using larger databases with different rates of outcomes may further clarify the differences between artificial neural network and logistic regression models in head injury outcome prediction and their clinical implications. Competing interests The author(s) declare that they have no competing interests. Authors' contributions BE carried out the data extraction, performed the analysis and drafted the manuscript. KM supervised the analysis and critically reviewed the statistical viewpoints. HEA, MG and EK supervised the study and participated in its coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551612.xml |
544579 | Phospholipids reduce gastric cancer cell adhesion to extracellular matrix in vitro | Background Nidation of floating tumour cells initiates peritoneal carcinosis and limits prognosis of gastro-intestinal tumours. Adhesion of tumour cells to extracellular matrix components is a pivotal step in developing peritoneal dissemination of intraabdominal malignancies. Since phospholipids efficaciously prevented peritoneal adhesion formation in numerous animal studies we investigated their capacity to reduce adhesions of gastric cancer cells to extracellular matrix components (ECM). Methods Human gastric cancer cells (NUGC-4, Japanese Cancer Research Resources Bank, Tokyo, Japan) were used in this study. Microtiter plates were coated with collagen IV (coll), laminin (ln) and fibronectin (fn). Non-specific protein binding of the coated wells was blocked by adding 1% (w/v) BSA (4°C, 12 h) and rinsing the wells with Hepes buffer. 50.000 tumour cells in 100 μl medium were seeded into each well. Beside the controls, phospholipids were added in concentrations of 0.05, 0.1, 0.5, 0.75 and 1.0/100 μl medium. After an incubation interval of 30 min, attached cells were fixed and stained with 0.1% (w/v) crystal violet. The dye was resuspended with 50 μl of 0.2% (v/v) Triton X-100 per well and colour yields were then measured by an ELISA reader at 590 nm. Optical density (OD) showed a linear relationship to the amount of cells and was corrected for dying of BSA/polystyrene without cells. Results The attachment of gastric cancer cells to collagen IV, laminin, and fibronectin could be significantly reduced up to 53% by phospholipid concentrations of 0.5 mg/100 μl and higher. Conclusion These results, within the scope of additional experimental studies on mice and rats which showed a significant reduction of peritoneal carcinosis, demonstrated the capacity of phospholipids in controlling abdominal nidation of tumour cells to ECM components. Lipid emulsions may be a beneficial adjunct in surgery of gastrointestinal malignancies. | Background In the treatment of gastro-intestinal cancer the detection of free, isolated tumour cells in the peritoneal cavity serve as a prognostic marker for postoperative survival [ 1 - 4 ]. Since surgery frequently proofs insufficient for tumour control, numerous additional treatments have been evaluated. A pivotal step in developing peritoneal dissemination seems to be adhesion of tumour cells to mesothelial cells or extracellular matrix components [ 5 - 7 ]. Experimental studies suggest that peritoneal metastases tend to occur in areas of injured peritoneum [ 8 ]. Cell-matrix interactions are promoted by transmembrane receptors with integrins as a major family. Many attempts were made to inhibit tumour cell attachment by antibodies against adhesion molecules [ 9 ], dextran sulphate [ 10 ], or sodium hyaluronate [ 11 ] with different results concerning tumour adhesion. Phospholipids, polar phosphoric acid di-esters, are natural constituents of the abdominal fluid. The substance is able to form a lubricant layer on the peritoneal surface [ 12 ]. Additionally, integrin function, particularly in control of cell motility is affected by exogenous addition of phospholipids (e.g. gangliosides) [ 13 , 14 ]. Intraperitoneal use of phospholipids (PL) led to a significant decrease of adhesion formation especially at sites of peritoneal injury [ 15 , 16 ]. The objective of the underlying in vitro study was focused on the influence of phospholipids on adhesion of gastric cancer cells to extracellular matrix components with broad reactivity to several integrins. Collagen IV (coll IV), and laminin (ln) are main components of the basement membrane and fibronectin (fn) plays an important role in wound healing [ 17 , 18 ]. Methods Tumour cells The human gastric cancer cell line NUGC-4 was purchased from the Japanese Cancer Research Resources Bank (Tokyo, Japan). The cells were maintained in monolayers in tissue culture flasks (75 cm 2 , Falcon, Becton Dickinson-Gambil, Heidelberg, Germany) in RPMI 1640 medium (GIBCO, Karlsruhe, Germany), supplemented with 10% foetal bovine serum (GIBCO), penicillin and streptomycin (GIBCO). Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO 2 in air. Cells were passaged after treatment with 0.125% trypsin for 6 min. The cells were pelleted after centrifugation for 10 min at 200 g, suspended in 20 ml PBS, and pelleted. The cell pellet was resuspended in 30 ml complete medium and seeded with a splitting ratio of 1:3. Only cells from three passages were used for the experiments. Extracellular matrix (ECM) components Flat-bottom polystyrene microtiter plates (Becton Dickinson, Heidelberg, Germany) were coated for adhesion experiments. The purified ECM components were dissolved in PBS with the following concentrations: coll IV – 2,5 μg/ml (Biomol, Hamburg, Germany), fn – 10 μg/ml (Boehringer, Mannheim, Germany), ln – 50 μg/ml (Boehringer, Mannheim, Germany). We found these concentrations to be optimal in foregoing dilution series. They were added to the wells and incubated at 4°C for 24 hours (coll IV, fn), or at 37°C for 45 min in a humidified atmosphere of 5% CO 2 in air (ln), respectively. Nonspecific protein binding of the coated wells was blocked by adding 1% (w/v) BSA (4°C, 12 h) and rinsing the wells with Hepes buffer. Adhesion assay For adhesion experiments gastric cancer cells were detached with collagenase I (15 min, 37°C, Worthington, Freehold, USA), washed once with RPMI 1640, centrifuged (200 g for 10 min), resuspended in RPMI 1640, and preincubated for 30 min in a humidified atmosphere of 5% CO2 in air (37°C). Fifty thousand tumour cells in 100 μl medium were seeded into each well. Evaluation of adherent cells was performed using crystal violet staining according to the method described by Aumeilley et al., and Tietze et al. [ 19 , 20 ]. After an incubation period of 30 min the supernatant with non-adherent cells was removed by two washes with warmed RPMI 1640. Attached cells were fixed with 30% (v/v) methanol/ethanol for 15 min at room temperature. Cells were stained with 0.1% (w/v) crystal violet (Sigma, Hamburg, Germany), extensively washed with distilled water, and dried at room temperature. The dye was resuspended with 50 μl of 0.2% (v/v) Triton X-100/well and colour yields were then measured using an ELISA reader at 590 nm (Titertek Multiscan Plus MKII, Flow Laboratories GmbH, Meckenheim, Germany). Optical density (OD) showed a linear relationship to the amount of cells between 1 × 10 3 and 5 × 10 4 cells per well, as determined by a dilution series. Control dying of BSA/polystyrene without cells led to Optical Density (OD) values of 0.01–0.07. These values were subtracted from those obtained in the experiments. Phospholipids After complete preparation of the tumour cell suspension, the PL solution was added in the following concentrations: 0.05, 0.1, 0.5, 0.75, and 1 mg per 100 μl medium. The concentrations used were correlated to our in vivo experiments. The phospholipid solution consists of phosphatidylcholine 70% by weight, phosphatidylethanolamine 15% by weight, neutral lipids 8% by weight, sphingomyelin <3% by weight and lysophosphatidylcholine <3% by weight. Statistical analysis All experiments were performed three times in quadruplicate. The data are expressed as means +/- standard error of the mean (SEM). Student's t-test for unpaired data was used for statistical analysis. Differences were regarded as significant for p values < 0.05. Results The analysis of tumour cell adhesion to BSA 1% resulted in a mean extinction of 0.27 (SEM 0.01) at 590 nm. Coating with ln and fn led to a nearly twofold increase of tumour cell adhesion with mean values of 0.59 (0.03, ln) and 0.63 (0.03, fn). The cancer cells showed a most pronounced adhesion to coll IV with a mean extinction of 0.97 (0.02). The tumour cell adhesion to ln registered after addition of PL was significantly reduced. The effect was concentration dependent compared to the controls. Even the minimum amount of PL 0.05 mg/100 μl led to a reduced extinction of 0.4 (0.01). Treatment with 0.1 or 0.5 mg/100 μl PL revealed extinction values of 0.32 (0.02) and 0.28 (0.02), respectively. The maximum effect could be demonstrated with 0.75 mg/100 μl PL with an extinction of 0.24 (0.02). The relative reduction of tumour cell adhesion compared to the control amounts to 59%. Treatment with 1 mg/100 μl PL showed no further decrease of tumour cell adhesion to ln. The mean extinction was 0.26 (0.01) (table 1 ). Table 1 Influence of different phospholipid concentrations on adhesion of gastric cancer cells to laminin. Optical density (OD) measured in an ELISA reader at 590 nm Extinction at 590 nm SEM p Control 0.59 0.03 PL 0.05 mg/well 0.4 0.01 p < 0.05 PL 0.1 mg/well 0.32 0.02 p < 0.05 PL 0.5 mg/well 0.28 0.02 p < 0.05 PL 0.75 mg/well 0.24 0.02 p < 0.05 PL 1 mg/well 0.26 0.01 p < 0.05 The tumour cell adhesion on fn could not be reduced significantly with low concentrations of Pl. Addition of 0.05 mg/100 μl PL and 0.1 mg/100 μl resulted in a slight reduction of the extinction with mean values of 0.59 (0.02) and 0.59 (0.01). However, a significant reduction of tumour cell adhesion could be observed after treatment with 0.5 mg/100 μl PL, 0.42 (0.02); as well as with 0.75 mg/100 μl PL (0.39 (0.02)) and 1 mg/100 μl PL (0.38 (0.02)). We found a similar situation compared to ln with equal effects of 0.75 mg/100 μl and 1 mg/100 μl PL indicating that the maximum influence on adhesion is reached. The relative reduction of tumour cell adhesion compared to the control values amounts to 40% (table 2 ). Table 2 Influence of different phospholipid concentrations on adhesion of gastric cancer cells to fibronectin. Optical density (OD) measured in an ELISA reader at 590 nm Extinction at 590 nm SEM p Control 0.63 0.03 PL 0.05 mg/well 0.59 0.02 n. s. PL 0.1 mg/well 0.59 0.01 n. s. PL 0.5 mg/well 0.42 0.02 p < 0.05 PL 0.75 mg/well 0.39 0.02 p < 0.05 PL 1 mg/well 0.38 0.02 p < 0.05 NUGC-4 gastric cancer cells prominently adhere to collagen IV compared to all other examined extracellular matrix components. The influence of PL on cell adhesion to coll IV was also concentration dependent. The reduction ranged from an extinction of 0.89 (0.01) after administration of 0.05 mg/100 μl PL to a maximum effect after treatment with 1 mg/100 μl PL with a value of 0.44 (0.02) (table 3 ). In comparison to the control value, this means a reduction of adherent tumour cells of 55%. Table 3 Influence of different phospholipid concentrations on adhesion of gastric cancer cells to collagen IV. Optical density (OD) measured in an ELISA reader at 590 nm Extinction at 590 nm SEM p Control 0.97 0.02 PL 0.05 mg/well 0.9 0.01 p < 0.05 PL 0.1 mg/well 0.74 0.02 p < 0.05 PL 0.5 mg/well 0.61 0.02 p < 0.05 PL 0.75 mg/well 0.45 0.03 p < 0.05 PL 1 mg/well 0.44 0.02 p < 0.05 Discussion Cell adhesion to the extracellular matrix plays a fundamental role in peritoneal carcinosis. The adhesion is mediated by transmembrane Integrins. Several proteins including fibronectin in the interstitial matrix, laminin and collagen IV in the basement membrane were identified as important ligands [ 21 , 22 ]. Many attempts were made to inhibit tumour cell adhesion by integrin antibodies or competitive inhibitors against specific peptide sequences [ 23 - 26 ]. Gui et al. showed that adhesion of different breast cancer cells to extracellular matrix components could be reduced by specific integrin antibodies [ 27 ]. However, different antibodies for different cell lines were necessary according to the expression of specific integrins on the cell surface. Haier et al. could find different adhesive capacities to collagen I in two subtypes of the HT-29 colon carcinoma cell line. The cells with a very limited capability to induce hepatic metastases showed a significant higher rate of adhesions compared to those inheriting a high potential for involvement of the liver [ 28 ]. The influence of three examined phosphotyrosine kinase inhibitors on integrin mediated tumour cell adhesion to collagen I was unspecific. Dennis et al. found different cell-surface receptors responsible for cell attachment to fibronectin and collagen as compared to laminin. They concluded with the hypothesis that specific glycolipids may be receptors for interaction with fibronectin [ 29 ]. In our experiments the reduced rate of cell attachment in the presence of phospholipids was independent from the extracellular matrix. A similar effect on intraperitoneal tumour growth was described by Jacobi et al. who could demonstrate that taurolidine/heparin and povidone iodine lead to a significant reduction of tumour cell growth in vitro as well as a reduction of tumour weight after intraperitoneal tumour injection [ 30 ]. Predominantly the result seems to be a attributed to the cytotoxic effect of the used substances and benefits to a lesser degree from adhesion prevention. Other substances used to prevent adhesion failed in the treatment of inhibiting tumour cell attachment. Sodium hyalunorate increased the metastatic potential of colo-rectal tumour cells, probably mediated by the CD44 receptor [ 31 ]. Dextran sulphate resulted in reduced tumour cell nidation at sites of injury to abdominal wall in mice [ 10 , 32 ]. However, several side effects were described in the use of dextrane for adhesion prevention. Main problems were oedema, pleura effusion, life-threatening coagulation disorders and severe allergic reactions [ 33 , 34 ]. Phospholipids, polar phosphoric acid di-esters, are natural constituents of the abdominal cavity fluid and cell membranes. The hypothesis is that phospholipids form a lubricant layer on the peritoneum by binding with its negatively charged cholin branch chain to the positively charged peritoneal surface [ 12 , 14 , 35 ]. Phospholipids cover the entire peritoneal membrane by a thin fluid layer. By separating tumour cells from the peritoneal surface they proved to significantly reduce peritoneal carcinosis. Phospholipipds seem to reduce the expression of integrins and adhesion molecules on the cell surface to the effect that adhesions can be prevented reducing tumour cell attachment independent from their origin [ 15 , 16 ]. The in situ tumour cell – ECM interaction is influenced by adhesive and non-adhesive ECM components and can be understood as a three dimensional network [ 36 ]. Therefore the in vitro experiments with tumour cells as soluble agents added to ECM immobilized onto plastic surfaces cannot appropriately mimic the situation in situ. Recently we found that phospholipids significantly reduce the attachment area and the tumour volume of peritoneal carcinosis caused by the colonic cancer cell line DHD/K12/TRb in rats. These results were supported by a prolonged survival rate of the treated animals as compared to the control group. Additionally, we found a similar effect of phospholipids on the adhesion of the human rectal cancer cell line HRT-18 on the same ECM-components in vitro [ 37 ]. Consistent with results of other groups, the tumour cell attachment was found predominantly in areas of previously injured peritoneum [ 5 , 6 , 38 , 39 ]. We performed this study to ascertain the results of the foregoing animal experiments and to demonstrate the influence of phospholipids to three different ECM components, even though matrices of collagen IV, laminin and fibronectin alone may not be predictive of peritoneal membrane nidation. Conclusion These results, within the scope of additional experimental studies on mice and rats which showed a significant reduction of peritoneal carcinosis, demonstrated the capacity of phospholipids in controlling abdominal nidation of tumor cells to ECM components. Lipid emulsions may be a beneficial adjunct in surgery of gastrointestinal malignancies. Competing interests The work was financially supported by Fresenius Kabi, Bad Homburg, Germany. The results are part of an international patent application. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544579.xml |
546409 | Evidence of connections between cerebrospinal fluid and nasal lymphatic vessels in humans, non-human primates and other mammalian species | Background The parenchyma of the brain does not contain lymphatics. Consequently, it has been assumed that arachnoid projections into the cranial venous system are responsible for cerebrospinal fluid (CSF) absorption. However, recent quantitative and qualitative evidence in sheep suggest that nasal lymphatics have the major role in CSF transport. Nonetheless, the applicability of this concept to other species, especially to humans has never been clarified. The purpose of this study was to compare the CSF and nasal lymph associations in human and non-human primates with those observed in other mammalian species. Methods Studies were performed in sheep, pigs, rabbits, rats, mice, monkeys and humans. Immediately after sacrifice (or up to 7 hours after death in humans), yellow Microfil was injected into the CSF compartment. The heads were cut in a sagittal plane. Results In the seven species examined, Microfil was observed primarily in the subarachnoid space around the olfactory bulbs and cribriform plate. The contrast agent followed the olfactory nerves and entered extensive lymphatic networks in the submucosa associated with the olfactory and respiratory epithelium. This is the first direct evidence of the association between the CSF and nasal lymph compartments in humans. Conclusions The fact that the pattern of Microfil distribution was similar in all species tested, suggested that CSF absorption into nasal lymphatics is a characteristic feature of all mammals including humans. It is tempting to speculate that some disorders of the CSF system (hydrocephalus and idiopathic intracranial hypertension for example) may relate either directly or indirectly to a lymphatic CSF absorption deficit. | Background The formation of CSF has been studied extensively [ 1 ] and the physical location at which it is formed within the ventricular system (the choroid plexus) has been well established. In contrast, while CSF absorption parameters have been investigated in many species, the mechanism and location of the transport sites is far from clear. It is assumed that projections of the arachnoid membrane into the cranial venous sinuses facilitate CSF removal however, there is very little convincing quantitative evidence to support a role for arachnoid villi and granulations in this process. Recent experiments suggest that the movement of CSF directly into the cranial venous system may only occur at high pressures suggesting that the arachnoid projections may have an accessory function rather than representing the primary locations at which CSF is absorbed [ 2 , 3 ]. The experimental evidence would seem to favour the view that a significant portion of CSF is removed from the subarachnoid space by nasal lymphatic vessels. This conclusion is based on experiments that span over 100 years of investigation [reviewed in [ 4 ]] with the most extensive analysis of CSF transport having been carried out more recently in sheep [ 2 , 3 , 5 - 17 ]. CSF convects through the cribriform plate into lymphatic vessels located in the submucosa associated with the olfactory and respiratory epithelium. The lymph within the ethmoidal lymphatics appears to be continuous with CSF in the perineurial spaces associated with the olfactory nerves. Multiple lymphatic ducts form a collar around the emerging nerve roots, which facilitates direct connection between CSF and lymph in the olfactory submucosa [ 18 ]. An extensive complex of lymphatic vessels in the ethmoid turbinal system conveys CSF through a variety of lymph nodes in the head and neck region to the cervical lymphatics that in turn, convey the CSF to the venous system [ 3 ]. There is also substantial data with various tracers and dyes that this lymphatic CSF transport pathway is a characteristic feature of many mammals [ 4 ]. However, there is still some uncertainty regarding the applicability of animal concepts to CSF transport in man. It has been argued that the larger brain, lack of sophisticated olfactory mechanisms and bipedal versus quadrapedal location make CSF transport in humans fundamentally different than that in animals. There is no easy way to address the issue of lymphatic CSF transport in humans but recent experience visualizing CSF-lymph connections with Microfil, suggested that it may be possible to examine lymphatic CSF transport pathways in any species post-mortem . Microfil is a coloured, liquid silicone rubber compound which, when combined with a catalytic curing agent, polymerizes within 20 minutes. This agent facilitates the generation of 3 dimensional images of the spaces into which it has been injected. In a previous report, we infused Microfil into the subarachnoid compartment of sheep and noted that it was carried through the cribriform plate into an extensive network of lymphatic vessels in the nasal submucosa of this species [ 2 , 3 , 17 , 18 ]. Drawing on this experience, the main objective of the studies reported here was to examine potential CSF-nasal lymph pathways in a variety of species including human and non-human primates. The results of this investigation support the view that lymphatics play an important role in CSF transport in all mammalian species. Methods Animals All animal experiments were approved by the ethics committee at Sunnybrook and Women's College Health Sciences Centre and conformed to the guidelines set by the Canadian Council on Animal Care and the Animals for Research Act of Ontario. Studies in the sheep, mouse, rat, rabbit and pig were performed at Sunnybrook and Women's College Health Sciences Centre. Studies on the Barbados green monkey were performed at the Barbados Primate Research Centre and Wildlife Reserve. Investigation of human specimens was approved by the Chief Coroner and the General Inspector of Anatomy for Ontario. These studies were conducted at the University of Toronto, Department of Surgery, Division of Anatomy. Experimental Objectives The objective of these studies was to infuse a CSF tracer, Microfil (Flowtech, Mass), into the cranial subarachnoid space post-mortem to outline the pathways of CSF outflow. To accomplish this, the specimen was placed in sternal recumbancy with the head fixed in position. A laminectomy was performed on the upper regions of the spinal cord (generally between C1-C2) and the cisterna magna was cannulated with an angiocatheter or a silastic tube of appropriate size. A ligature was placed around the spinal cord and tightened to compress the spinal cord and meninges [ 13 ]. This ligature served two purposes. First, as we were interested in cranial absorption pathways, the amount of Microfil infused was less since this agent was prevented from passing into the spinal subarachnoid compartment during injection. Second, our experience suggested that infusion pressures effective in filling the pathways of interest were more easily achieved with the spinal CSF space negated. With the exception of studies in the mice, all experiments were performed by infusing Microfil manually using a syringe. We did not routinely monitor infusion pressures post-mortem. However, in several of our initial studies in sheep, we measured infusion pressures between 200 and 300 cm H 2 O. Mice on the other hand appeared to be very fragile and injection by hand was problematic. In this species, we found that Microfil filling was more successful if this agent was introduced into the CSF compartment using a free-flow pressure system and reservoir as described below. The best results were obtained using a Microfil preparation that was more dilute than that recommended in the product literature. To achieve this, 3 ml of diluent was used for every 1 ml of yellow Microfil ® (MV-122) and the material catalyzed with 10% (of total volume) of MV Curing Agent. In several sheep, blue Microfil (prepared as recommended in the instruction manual) was introduced into the vasculature at the same time that yellow Microfil was injected into the CSF compartment. The carotid arteries were catheterized and 20 ml of blue Microfil ® (MV-120) was infused into both arteries simultaneously. More specific details pertaining to each species is outlined below. Mice Randomly bred, wild-type C57/Bl mice ( n = 21, 16–19 gm; ~4–5 weeks old) were used for this investigation. They were fed lab rat chow (LabDiet 5001) until sacrifice. At the start of the preparation, the mice were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space by one of two methods. In some preparations, Microfil was infused into the cranial subarachnoid space over a 5–10 minute interval through manual injection into an angiocatheter. In other experiments, the Microfil was administered using a free-flow pressure system with a reservoir filled with saline regulating the rate of flow into the cisterna magna. To achieve this, the three ports of a 3-way stopcock were attached to (a) a syringe containing Microfil, (b) a cannula attached to an angiocatheter inserted into the subarachnoid space, and (c) a cannula filled with saline hung vertically from a pole. The reservoir height was adjusted such that the saline solution exerted a pressure of approximately 100 cm H 2 0. Microfil was injected via the syringe until the agent reached the hub of the angiocatheter. At this point, the stopcock was adjusted to allow the column of PBS to exert a pressure on the Microfil, which pushed the contrast agent into the subarachnoid space. Rats Randomly bred, Fischer 344 rats ( n = 6, ~300 gm; 11–15 weeks old) were used for this investigation. They were fed lab rat chow (LabDiet 5001) until sacrifice. The rats were anaesthetized initially by placement in an induction chamber in which 5% halothane was administered for 2 minutes. Immediately after anesthesia, the rats were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space over 5–10 minutes. Rabbits Randomly bred, New Zealand white rabbits ( n = 2, 3 and 4 kg; ~18 weeks old) were used for this investigation. They were fed rabbit chow (LabDiet 5321) until sacrifice. The rabbits were initially anesthetized with an intramuscular injection of Ketamine (50 mg/kg) and Rompun (5 mg/kg). Prior to surgery, the rabbits were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space over a 5–10 minute period. Sheep Randomly bred sheep ( n = 29; 2.6–35 kg) including newborns (2–7 days old) and adult animals (6–8 months old) were used for this investigation. The neonatal lambs were bottle-fed formula (Lamb Replacement, Grober Inc. Cambridge, Ontario) until surgery. The lambs were anaesthetized initially by mask administration of incremental concentrations of halothane from 0.5 to 3%. The adult sheep were anesthetized initially by intravenous infusion of 2.5% sodium Pentothal solution. Subsequently, 1–3% halothane was delivered through an endotracheal tube via an A.D.S.1000 or Narkomed 2 respirator for surgical maintenance. In this species, a laminectomy was performed at the C1-C2 level under anesthesia followed by sacrifice. Microfil was infused into the cranial subarachnoid space over 10–15 minutes. In some animals, blue Microfil (prepared as recommended in the instruction manual) was introduced into the vasculature at the same time that yellow Microfil was injected into the CSF compartment. The carotid arteries were catheterized and 20 ml of blue Microfil ® (MV-120) was infused into both arteries simultaneously. Pigs Randomly bred, Yorkshire pigs ( n = 5, 20–25 kg; 8–9 weeks old) were used for this investigation. They were fed pellets (LabDiet Laboratory Animal Diet) until sacrifice. The pigs were anesthetized initially with an intramuscular injection of Ketamine (30 mg/kg) and Atropine (0.04 mg/kg). Surgical anesthesia was induced and maintained by 2–5% halothane delivered through an endotracheal tube via a Narkomed 2 respirator. Prior to surgery, the pigs were euthanized. A laminectomy was performed at the C1-C2 level and Microfil was infused into the cranial subarachnoid space over a 10–15 minute period. Monkeys Randomly bred, Barbados green monkeys ( Cercopithecus aethiops sabeus ) ( n = 6, 4.0–5.6 kg; ~6 years old) were used for this investigation. They were fed twice daily various natural foods until sacrifice. At the start of the preparation, the monkeys were anaesthetized with Xylazine/Ketamine (100 mg/kg) injection. Subsequently, the monkeys were euthanized. A laminectomy between C7 and T1 was performed and Microfil was infused into the cranial subarachnoid space over a 5–10 minute period. Humans Three recently deceased cadaveric preparations were examined (3 females, 77, 80 and 90 yrs old). Access to the cadaveric material was permitted between 6 and 7 hours post mortem. A laminectomy between C7 and T1 was performed and Microfil was infused into the cranial subarachnoid space. Analysis of tissues Following injection with Microfil, the heads of mice, rats, rabbits, sheep and pigs were stored at 4°C overnight. The next day, the heads were skinned, sectioned in a sagittal orientation and fixed in 10% formalin. In the monkey studies, the Microfil was allowed to set for 2 hours after injection followed by sectioning and fixation with formalin (2–3 weeks) and 70% alcohol (immediately before shipment to Canada). After Microfil infusion in humans, the heads were placed in a freezer for several days (-18°C) after which they were sectioned and fixed with Kaiserling's solution for at least 5 days before dissection. Sectioning of the fixed tissues was made in several orientations including coronal cuts anterior and posterior to the cribriform plate and/or sagittal sections. The various tissues were dissected as appropriate under a dissecting microscope (Leica M651, Wild Leizt; EMZ-TR, Meiji Techno; Fisher Stereomaster) and images were captured on a Nikon digital camera (Coolpix 995). All images presented in this report are in the midsagittal plane from the cut surfaces of the heads. Results Technical Issues Filling of the subarachnoid compartment and extension of the Microfil into the lymphatic vessels of the olfactory and respiratory submucosa was dependent on several factors including the size of the species and the time of injection after death. Filling of the CSF space and attendant lymphatic vessels with Microfil was easiest in the larger animals and was increasingly challenging as the species declined in size. Additionally, we were able to achieve successful filling up to 6 hours after death but the best results were obtained when the agent was injected immediately after the animal was euthanized. For reasons unknown, in a few unsuccessful preparations the Microfil did not cure correctly. The number of human specimens was limited to 3. In one attempt, there was incomplete filling of the subarachnoid space due to rupture of the meninges and dura. In another preparation, a large metastatic tumor located in the frontal lobe with accompanying perifocal edema and petechial hemorrhages caused significant dislocation of the brain and prevented filling of the CSF spaces. In a third preparation, where the cause of death was not connected to brain disease (lung cancer), the infusion was successful. Table 1 summarizes the experimental details and the success rates of the Microfil infusions in the various species. Table 1 Details of animal studies Species Number of animals used Number of successful preparations Hours after death Injected Microfil volume (ml) mice 21 7 0–1 1–1.5 rats 6 4 0–1 2.5–3 rabbits 2 2 0–1 10 sheep 29 26 0–1 20–25 pigs 5 2* 2–72 20–25 monkeys 6 5 0–1 20 humans 3 1** 5–7 60–180 * Successful filling was achieved 2 and 5 hours after death ** In the one successful human preparation, 180 ml of Microfil was infused CSF Transport Pathways Microfil was observed throughout the subarachnoid compartment associated with the base of the brain and generally filled the basal cisterns in all species. Microfil was also observed commonly in the subarachnoid space over the convexities of the brain. In all species examined in this report (mouse and rat, Figure 1 ; rabbit and sheep, Figure 2 ; pig and monkey, Figure 3 ; and human, Figure 4 ), Microfil could be observed in a patchy distribution along the olfactory nerves external to the cranium and in lymphatics associated with the submucosa of the olfactory epithelium. More extensive lymphatic networks containing Microfil were observed in the submucosa of the ethmoid labyrinth and adjacent nasal septum. In some of the sheep preparations, blue Microfil was introduced into the vascular system and yellow Microfil into the subarachnoid space. Yellow lymphatic vessels were easily distinguished from blue arterioles (Figure 5A ). Figure 1 Microfil distribution patterns in the head of mice and rats. All images are presented in sagittal plane with gradual magnification of the olfactory area adjacent to the cribriform plate. Reference scales are provided either as a ruler in the image (mm) or as a longitudinal bar (1 mm). A-C illustrates images of the mouse and D-F images of rat. The Microfil can be viewed in the perineurial space of the olfactory nerves external to the cranium and a network of lymphatic vessels containing yellow Microfil can be observed in the ethmoid turbinal systems of both species. In the example illustrated in 1C, the image was captured before fixation. The reddish spots are areas of hemorrhage in this particular animal. b – brain; cp – cribriform plate; et – ethmoid turbinates; ob – olfactory bulbs; on – olfactory nerves; ns – nasal septum. Figure 2 Microfil distribution patterns in the head of rabbits and sheep. All images are presented in sagittal plane with gradual magnification of the olfactory area adjacent to the cribriform plate. Reference scales are provided either as a ruler in the image (mm) or as a longitudinal bar (1 mm). A-C illustrates images of the rabbit and D-F images of sheep. In both species, the Microfil can be viewed in the perineurial spaces of the olfactory nerves external to the cranium, which merge into network of lymphatic vessels in the ethmoid turbinal systems. b – brain; cp – cribriform plate; et – ethmoid turbinates; on – olfactory nerves; ns – nasal septum; arrow in D – portion of cribriform plate removed for histology. Figure 3 Microfil distribution patterns in the head of pigs and Barbados green monkeys. All images are presented in sagittal plane with gradual magnification of the olfactory area adjacent to the cribriform plate. Reference scales are provided either as a ruler in the image (mm) or as a longitudinal bar (1 mm). A-C illustrates images of the pig and D-F images of the monkey. In both species, the Microfil can be viewed in the perineurial spaces of the olfactory nerves external to the cranium, which merge into network of lymphatic vessels in the ethmoid turbinal systems. In A, Microfil can be observed penetrating the dura and entering the cavernous sinus with partial filling of the retromandibular vein (arrow). b – brain; cp – cribriform plate; et – ethmoid turbinates; ob – olfactory bulbs; on – olfactory nerves. Figure 4 Microfil distribution patterns in the head of a human (A-F). All images are presented in sagittal plane with gradual magnification of the olfactory area adjacent to the cribriform plate. Reference scales are provided either as a ruler in the image (mm) or as a longitudinal bar (1 mm). As in the other species, Microfil introduced into the subarachnoid space was observed around the olfactory bulb (A), in the perineurial spaces of the olfactory nerves (B, C) and in the lymphatics of the nasal septum (D), ethmoid labyrinth (E) and superior turbinate (F). Due to tissue deterioration, some of the lymphatic vessels had ruptured and Microfil was noted in the interstitium of the submucosa of the nasal septum (D). In (E), Microfil is observed in the subarachnoid space and the perineurial space of olfactory nerves. The perineurial Microfil is continuous with that in lymphatic vessels (arrows). Intact lymphatic vessels containing Microfil are outlined with arrows (D, E, F). b – brain; fs – frontal sinus; cp – cribriform plate; et – ethmoid turbinates; ob – olfactory bulbs; on – olfactory nerves; ns – nasal septum; sas – subarachnoid space. Figure 5 Visualization of lymphatic vessels containing Microfil. Reference scales are provided as a longitudinal bar (1 mm). A – illustrates the ability to separate blood vessels (blue) from lymphatics (yellow) using differently coloured Microfil preparations (sheep). B – demonstrates lymphatic networks that have taken up Microfil that was injected into the subarachnoid compartment (pig). These networks ultimately connect with various lymph nodes (C – retropharyngeal node example in sheep; D – submandibular node example in mouse). The prenodal vessels can be visualized converging on the lymph nodes with the lymphatics congregating into a labyrinth of small ducts or foot processes on the node capsule. Blood vessels containing blue Microfil can be observed proximal to the node in C. The functional contractile unit of lymphatic vessels is the lymphangion, which is a segment of vessel between 2 valves (valves illustrated by arrows in C). n – lymph node; L – lymphangion. The structural properties of the ethmoid labyrinth determined the density of distribution of Microfil into lymphatics of the nasal cavity. The rat, mouse, rabbit, sheep, and pig have large olfactory bulbs and a broad, deep olfactory fosse in the cribriform plate. The structure of the ethmoid bone determines the broad distribution of olfactory nerves from the midline to the latero-posterior nose. In monkeys and humans, the ethmoid bone is more delicate and the olfactory nerves are concentrated along the midline. The exits of olfactory nerves from the skull base were filled with Microfil and in most successful preparations it was possible to see the trunks of the olfactory nerves with Microfil distributed in the perineurial space. We reported previously that Microfil was observed primarily within lymphatic vessels in the sheep with very little evidence of this agent within the interstitium of the submucosa [ 17 , 18 ]. While we did not investigate the tissues with histological techniques in this report, this also seemed to be the case in all of the species examined except for the human specimens. This was most likely due to the fact that we were able to inject the Microfil immediately or shortly after death in the animals. In humans however, there was a delay in getting access to the cadavers with the result that there was some degree of deterioration of the tissues. From previous studies in which Microfil was injected into animals at various times after sacrifice, we know that the CSF transport pathways into lymphatics degenerate rapidly after death and the filling of the vessels with Microfil is compromised. In any event, while many vessels had ruptured in the human tissues, Microfil was observed in enough intact lymphatic vessels to confirm the principle of CSF-nasal lymph connections in man. It has been well documented that CSF transports through the cribriform plate into nasal lymphatics which progress downstream to various lymph nodes including retropharyngeal, cervical, submandibular and preauricular lymph nodes. In some preparations in sheep and mice we were able to follow Microfil-containing lymphatic networks to the point at which they converged on these lymph nodes (examples are illustrated in Figure 5C,5D ). Some Microfil was observed in various veins in the head and neck region external to the cranium. An example can be seen in Figure 3A in the pig. In this case the retromandibular vein was partially filled with Microfil. Additionally, in some cases, Microfil was observed to enter the venous sinuses within the cranium. In some of the preparations in all species, Microfil was observed lying freely in the nasal cavity. This material lay proximal to terminal branches of the olfactory nerves and supports the concept of physiological and post inflammatory CSF rhinorrhea. Discussion Nasal lymphatic vessels have been implicated in CSF transport for many years. However, the concept has not received mainstream acceptance for several reasons. First, the data supporting lymphatic function has been based entirely on animal studies especially the sheep. However, histological and radiological evidence indicate that CSF-lymph connections exist in other species including rats [ 8 , 19 - 24 ], mice [ 25 ], rabbits [ 26 - 28 ], cats [ 20 , 29 , 30 ], dogs [ 20 , 30 ], guinea pigs [ 31 ], and non-human primates [ 30 , 32 ]. In the latter case, injection of radioactive albumin into the CSF compartment of monkeys led to elevated concentrations of tracer in the cervical lymph nodes [ 32 ]. Circumstantial evidence also suggests that similar CSF-lymphatic connections exist in humans as well [ 22 , 33 - 36 ]. For example, Indian ink administered into the CSF in human autopsy material [ 35 ] was observed to fill the perineurial spaces around the olfactory nerve branches and was found in the nasal submucosal tissue. Similarly, in an individual with subarachnoid hemorrhage, red blood cells were observed around the olfactory nerves and within the nasal mucosa. However, until now, direct evidence that the CSF and lymph compartments were linked in human and non-human primates was lacking. The Microfil images presented in this report demonstrate that the passage of CSF along the olfactory nerves into lymphatics is a characteristic feature of several mammalian species. The Microfil distribution patterns from the mouse to the human were remarkably similar. It must be acknowledged that we had limited access to human material and that studies with the cadaveric material are ongoing. However, the fact that Microfil was observed external to the cranium in the nasal submucosa adjacent to the cribriform plate in both human and Barbados green monkey specimens supports the view that CSF absorption in primates is not significantly different (at least qualitatively) from that in other mammalian species. In a previous study, we injected yellow Microfil into the subarachnoid space and blue Microfil into the carotid arteries [ 18 ]. This protocol permitted the separation of the blood vessels from lymphatics without the use of promiscuous molecular markers. Indeed, the larger Microfil-filled lymphatic vessels demonstrated the classical 'lymphangion' structure with constrictions of the vessels where the valves were positioned (example in Fig 5C ). The vessels could be traced in some instances to prenodal ducts that converged on various lymph nodes [ 18 ]. In the study reported here, we have included examples of this in sheep and mice (Figure 5C,5D ). Studies with Microfil provide valuable information on 'potential' CSF transport routes but one must be cautious in applying post-mortem observations to physiological processes. However, in sheep there is considerable quantitative data that supports the concept that the cribriform-olfactory nerve-lymphatic pathway contributes significantly to volumetric CSF absorption [ 2 , 3 , 5 - 9 , 11 , 13 - 16 ]. Given that the Microfil distribution patterns in sheep, pigs, rabbits, rats, mice, Barbados green monkeys and humans appear remarkably similar, it would seem likely that this pathway is important for CSF clearance in each of these species. Another reason for the skepticism regarding the role of lymphatics in CSF transport is the generally held view that arachnoid projections function in this role. However, the quantitative evidence supporting a role for arachnoid projections is very limited and unconvincing [ 37 - 39 ]. It must be acknowledged that we observed Microfil within a variety of veins external to the cranium and in the dural sinuses. With regards to the former, Microfil entry was due (at least in part) to passage of the contrast agent through the dura at the base of the brain into the cavernous sinus. The Microfil appeared to push into this venous system and fill several veins. Whether this observation has any physiological relevance is unknown. The route by which Microfil gains entry into the dural venous sinuses is unknown but is currently under investigation. However, the concept of direct CSF transport into the superior sagittal sinus continues to be problematic. We used several experimental approaches to assess this issue in previous studies in sheep. The most informative technique was to employ the classical approach of Mann to look for enrichment of a radioactive CSF tracer in the superior sagittal sinus compared to a peripheral vein [ 40 ]. The Mann group observed that the concentration of soluble and particulate tracer was considerably greater in the sinus blood than in the peripheral circulation (inulin concentrations were up to 30 times greater). However, the peak CSF pressures achieved during bolus infusions were greater than 80 cm H 2 O, a level of pressure that is clearly pathological. Other groups have failed to observe enrichment of a CSF tracer in the superior sagittal sinus of the rabbit, cat and monkey when the intracranial pressures achieved in the experiments simulated physiological conditions more realistically [ 32 , 41 , 42 ]. In our investigations in adult sheep, at a CSF-superior sagittal sinus pressure gradient of around 10 cm H 2 O (normal gradient ~5 cm H 2 O) no enrichment was observed. When the gradient favouring absorption was greater than 20 cm H 2 O, a modest enrichment of tracer was noted [ 3 ]. A similar result was obtained with tracer enrichment experiments in newborn lambs. The latter result was surprising. Adult sheep have abundant arachnoid projections in the superior sagittal sinus which are visible when stained with Evans blue dye but, in the neonatal lamb, arachnoid projections are few or absent [ 2 ]. Therefore, tracer transport into the superior sagittal sinus could not be correlated with the presence or absence of arachnoid projections although some transport directly into the cranial venous system seemed to occur. In our studies the maximum enrichment of CSF tracer in the superior sagittal sinus was observed only within the first few minutes after increasing intracranial pressure [ 3 ]. Within a short time, the tracer concentrations in the sagittal sinus and peripheral venous blood were equal. This suggested the possibility that the tracer enrichment method outlined above has the potential to underestimate the role of arachnoid projections since vigorous tracer transport to peripheral veins by some other mechanism could cancel out any perceived tracer enrichment in the cranial venous sinuses. Projections of the arachnoid membrane into the veins at the base of the brain have been described but at this time, there is no evidence that these structures play a role in CSF transport. The most obvious contributor to the peripheral venous tracer would be the lymphatic circulation. To determine if the lack of CSF tracer enrichment in the superior sagittal sinus was due to lymphatic transport into peripheral veins, we created what we thought would be the conditions most favourable to measure clearance via the arachnoid granulations. We sealed the cribriform plate and prevented CSF transport into the spinal subarachnoid compartment thus negating much of the lymphatic transport capacity. Nonetheless, even under these conditions when the CSF-superior sagittal sinus pressure gradient was raised to 20 cm H 2 O (~4 fold higher than normal), no enrichment was observed. At pressure gradients greater than this level, the concentration of the tracer was significantly higher than in the peripheral circulation. Therefore, the lack of enrichment of the CSF tracer in the superior sagittal sinus at normal physiological intracranial pressures does not appear to be due to downstream lymphatic transport of the CSF tracer. Contrary to the 'classical' view, direct transport of CSF into cranial veins (whether this occurs through arachnoid projections we cannot determine unequivocally) may represent an auxiliary system that is recruited when intracranial pressures are high or when other transport pathways are compromised. In summary, we have never been able to obtain persuasive evidence that direct CSF transport into the venous system is a physiological reality. In contrast, the qualitative and quantitative support for nasal lymphatic role in CSF absorption is gaining momentum. Conclusions In this report, we present the first direct evidence for CSF-lymph connections in primates including monkeys and one human. The fact that the Microfil distribution patterns were very similar in the seven species tested suggested that the lymphatic transport of CSF represented an important pathway by which CSF is cleared from the subarachnoid space in mammalian species. A workable hypothesis can be developed around the concept that defects in lymphatic CSF transport may contribute to syndromes characterized by elevated intracranial pressure and/or hydrocephalus. Competing Interests The author(s) declare that they have no competing interests. Author's Contributions MJ: conceived of the study, and participated in its design and coordination. AZ: developed the application of the Microfil method to the analysis of CSF transport pathways, participated in all experiments, created an image catalogue and helped in the preparation of the manuscript. CP: aided in the experiments in human, sheep, rats and mice and helped in the preparation of the manuscript GS: performed the studies in rats and mice DA: in conjunction with AZ performed the studies in humans and monkeys and assisted in all experiments. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546409.xml |
546408 | Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis | Background Antibodies to a cytosolic soluble liver antigen (SLA) are specifically detected in patients with autoimmune hepatitis (AIH). The target of anti-SLA has been identified as a ~50 kDa UGA serine tRNA-associated protein complex (tRNP (Ser)Sec ), through the screening of cDNA libraries. A recent report questioned the identity of tRNP (Ser)Sec as the real SLA antigen. The latter study identified α-enolase as a major anti-SLA target, through proteomic analysis. Methods In an attempt to explain the observed discrepancy we have investigated reactivity of SLA positive sera against α-enolase and tRNP (Ser)Sec using rat and primate liver homogenate and the recombinant antigens. Thirty-three serum samples, 11 from SLA-positive patients and 22 from SLA negative controls were investigated. SLA antibodies were detected by an inhibition ELISA and confirmed by immunoblot using human liver homogenate. Autoantibody reactivity was further evaluated using preparations of primate and rat liver homogenates. Anti-α-enolase antibody reactivity has been tested by immunoblot using recombinant α-enolase. An affinity purified goat polyclonal anti-α-enolase IgG antibody was used as reference serum sample. Anti-tRNP (Ser)Sec antibody reactivity was detected by ELISA or dot blot using recombinant tRNP (Ser)Sec antigen. Results and Discussion The affinity purified IgG antibody directed to human α-enolase gave a band of approximately 48 kDa in both human and rat liver homogenates. A high titre anti-tRNP (Ser)Sec antibody serum gave a single band of ~50 kDa in both liver preparations. All but one anti-SLA antibody positive sera reacted with a ~50 kDa but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recombinant full length tRNP (Ser)Sec protein. None of the anti-SLA negative sera reacted with tRNP (Ser)Sec . Anti-SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot. Pre-incubation of anti-SLA positive sera with tRNP (Ser)Sec completely abolished the 50 kDa band. The findings of the present study indicate that α-enolase and tRNP (Ser)Sec are both expressed in primate and rat liver and have a respective MW of 48 and 50 kDa. They also show that anti-tRNP (Ser)Sec – but not anti-α-enolase – correlates with anti-SLA antibody reactivity. Conclusion Our findings indicate that tRNP (Ser)Sec is the most likely target of anti-SLA. | Background Antibodies to a cytosolic soluble liver antigen (SLA), detected originally by an inhibition ELISA using cytosolic liver fractions in a sub-group of patients with autoimmune hepatitis (AIH) negative for other autoantibodies, have recently been also reported in adult patients with anti-nuclear and/or smooth muscle antibody (ANA/SMA) positive type 1 AIH and in seronegative patients with a form of cryptogenic hepatitis resembling type 1 AIH [ 1 - 6 ]. In pediatric patients, anti-SLA has been described not only in type 1 AIH but also in anti-liver kidney microsomal-1 antibody positive type 2 AIH and autoimmune sclerosing cholangitis [ 7 - 10 ]. Anti-SLA is specific for these autoimmune liver diseases, where it is associated with a more severe course and is virtually absent in non-hepatic autoimmune disorders [ 1 - 9 ]. The target of anti-SLA has been identified by several groups as a ~50 kDa UGA serine tRNA-associated protein complex (tRNP (Ser)Sec ), through the screening of cDNA libraries [ 2 - 4 , 7 ]. Anti-tRNP (Ser)Sec antibodies have been detected in up to 90% of serum samples positive for SLA by the original inhibition ELISA [ 1 - 8 ]. Using anti-SLA positive sera against rat liver cytosolic fraction in one and two-dimensional immunoblotting analyses and through peptide mass fingerprint analysis, following MALDI-TOF mass spectrometry, Ballot et al. [ 11 ] identified four isoforms of α-enolase, – a cytosolic antigen of 48–50 kDa –, as the major target of anti-SLA positive sera. These findings challenge the notion that tRNP (Ser)Sec is the sole target of anti-SLA antibodies [ 2 - 8 ]. Critically, no absorption studies were performed with purified α-enolase to confirm this proposal [ 11 ]. Moreover, α-enolase has been described as an antigen in several autoimmune disorders totally unrelated to autoimmune hepatitis [ 12 - 18 ]. Using recombinant tRNP (Ser)Sec antigen as competitor in inhibition experiments it has been found removal of the 50 kDa band immunofixed by SLA positive sera from immunoblots of primate liver homogenate [ 19 ]. Though this finding indicates tRNP (Ser)Sec as a major component of SLA, a view apparently shared by Ballot et al, several questions still remain unanswered: 1. Are there any differences in α-enolase expression between rat – used by Ballot et al [ 11 ] – and primate liver homogenate – used by our study [ 19 ] – that could explain the discrepancy between these studies? 2. Is it true that failure of proteomic analysis to detect tRNP (Ser)Sec is due to its presence in trace amounts in the supernatant of liver homogenate [ 11 ]? 3. What is the reactivity of SLA positive and negative sera against recombinant α-enolase? 4. How do we explain the apparent paradox of SLA being identified as α-enolase by proteomic analysis and as tRNP (Ser)Sec by the screening of cDNA libraries? Do α-enolase and tRNP (Ser)Sec cross-react? In the present study, we have investigated reactivity of SLA positive sera against α-enolase and tRNP (Ser)Sec using rat and primate liver homogenate and the recombinant antigens. Methods Patients Thirty-three serum samples, 11 from SLA-positive patients and 22 from SLA negative controls were investigated. SLA-positive patients included 8 paediatric patients with AIH1 [7 female, median age 12, range 5–17 years, all ANA positive, median immunofluorescence (IFL) titre: 1/320, range 1/80–1/1280] and 3 adults with AIH/PBC overlap syndrome, (2 female, median age 56, range 47–65), all but one AMA positive (1/5120), and ANA positive (median tire 1/320, range 1/80–1/640). Eleven case-matched SLA negative patients were tested as pathological controls including 8 with AIH1 and 3 with AIH/PBC overlap syndrome. Eleven demographically matched healthy subjects including 8 children (7 female, median age 11, range 6–16) and 3 adults (2 female, median age 53, range 42–63) negative for SLA were also tested as controls. Antibody Detection All sera have been tested for conventional antibodies by indirect IFL using rodent liver, kidney, stomach tissues. SLA antibodies were detected by a modified inhibition ELISA [ 1 ] and confirmed by immunoblot using human liver homogenate [ 20 ]. Autoantibody reactivity was further evaluated using preparations of primate (Euroimmun, Lubeck, Germany) and rat (AID Autoimmun Diagnostika GmbH, Strassberg, Germany) liver homogenates, according to manufacturers' instructions. Anti-α-enolase antibody reactivity has been tested by immunoblot using recombinant α-enolase, as described previously [ 17 ]. Briefly, the complete complementary DNA (cDNA) encoding human α-enolase was isolated from a cDNA expression library derived from synoviocytes obtained from a patient with rheumatoid arthritis (Stratagene, La Jolla, CA) and immunoscreened with goat anti-enolase antibodies. This cDNA was subcloned in frame in the pSPUTK in vitro translation vector (Stratagene) using the Apa I and Bam HI restriction sites. The translation product was synthesized as a separated biotinylated polypeptide, purified by SoftLink Soft Release Avidin Resin (Promega, Madison, WI), migrated in SDS-PAGE, according to the method of Laemmli, and electrotransferred onto a nitrocellulose membrane [ 17 ]. The filters were then incubated with goat anti-enolase antibodies (see below), a monospecific anti-α-enolase antibody positive serum from a patient with rheumatoid arthritis or with individual serum samples, in Tris buffered saline, 0.05% Tween 20, 5% dry milk for 2 hours. After washing, the filters were incubated for 1 hour with 1:15,000-diluted peroxidase-conjugated goat anti-human IgG (Sigma-Aldrich) in 0.05% TBST-milk. The filters were washed and revealed by a chemiluminescence reaction (Supersignal; Pierce, Rockford, IL) [ 17 ]. An affinity purified goat polyclonal anti-α-enolase IgG antibody raised against a peptide mapping near the carboxyl-terminus of human α-enolase, which is common to α, β, and γ isoforms of mouse, rat and human enolase (200 μg/ml; Santa Cruz Biotechnology, Santa Cruz, California, USA) was used as reference serum sample at a dilution of 1:100, according to the manufacturer's instructions. Anti-tRNP (Ser)Sec antibody reactivity was detected by ELISA or dot blot using recombinant tRNP (Ser)Sec antigen (Euroimmun). A high-titre anti-tRNP (Ser)Sec antibody positive serum was used as a positive control. Inhibition Studies To investigate whether the 50 kDa band immunofixed by anti-SLA is tRNP (Ser)Sec , inhibition experiments were performed using 3 anti-SLA positive serum samples, diluted at 1/1000, and pre-incubated with solid phase recombinant tRNP (Ser)Sec (Euroimmun, UK), as previously described [ 19 ]. Results and Discussion The affinity purified IgG antibody directed to human α-enolase gave a band of approximately 48 kDa in both human and rat liver homogenates as shown in Figure 1 . A high titre anti-tRNP (Ser)Sec antibody serum gave a single band of ~50 kDa in both liver preparations (Figure 1 ). All but one anti-SLA antibody positive sera reacted with a ~50 kDa band similar to that obtained by the high titre anti-tRNP (Ser)Sec antibody serum in rat liver preparations (Figure 2 ) but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recombinant full length tRNP (Ser)Sec protein both in ELISA (mean titre 87 ± 23 RU/ml, cut off: 20 RU/ml) and dot blot. None of the anti-SLA negative sera reacted with tRNP (Ser)Sec . Anti-SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot with 5/9 cases in each group giving a strong band (Figure 3 ). Figure 1 Immunoblot patterns produced by anti-α-enolases and anti-tRNP (Ser)Sec antibodies on electrophoretically separated primate and rat liver homogenates and dot blot results with recombinant tRNP (Ser)Sec . In both rat and primate liver preparations, a band of ~48 kDa is immunofixed by a polyclonal goat IgG anti-α-enolase specific antibody; a band of ~50 kDa is immunofixed by a serum containing a high-titre anti-tRNP (Ser)Sec antibody. Anti-α-enolase antibody does not recognize tRNP (Ser)Sec by dot blot analysis. Figure 2 Immunoblot patterns produced on rat liver homogenate by (lane 1) a polyclonal goat IgG anti-α-enolase specific antibody; (lanes 2–5) four representative anti-soluble liver antigen (SLA) positive sera; (lane 6) a reference serum containing high-titre anti-tRNP (Ser)Sec antibody. Figure 3 Immunoblot patterns obtained using 9 SLA positive and 9 SLA negative serum samples against recombinant α-enolase. A polyclonal goat IgG anti-α-enolase specific antibody has been used as a reference positive serum. Ab, antibody; ag, antigen Pre-incubation of anti-SLA positive sera with tRNP (Ser)Sec completely abolished the 50 kDa band immunofixed in either rat or human liver preparation. In contrast, a parallel experiment where the anti-α-enolase antiserum was pre-incubated with recombinant tRNP (Ser)Sec left unaltered the reactivity to the 48 kDa band. The findings of the present study indicate that α-enolase and tRNP (Ser)Sec are both expressed in primate and rat liver and have a respective MW of 48 and 50 kDa. They also show that anti-tRNP (Ser)Sec – but not anti-α-enolase – correlates with anti-SLA antibody reactivity suggesting that the target of anti-SLA antibody is tRNP (Ser)Sec and not α-enolase [ 1 - 9 , 19 ]. However, Ballot et al [ 11 ] state that α-enolase is a major SLA antigen since rat α-enolases have MW of 47.4 to 47.5 and Pi values of 5.8 to 6.2. These characteristics do not match the MW (48.8) and Pi (8.6) of tRNP (Ser)Sec . Ballot et al [ 11 ], however, show a Coomassie stained 2D-gel over the Pi range 6 to 11 and an immunoblot of this gel with several bands of a Pi above 8, but have not investigated these bands by MALDI-TOF analysis being therefore unable to rule out their possible relation to tRNP (Ser)Sec . Conclusion All the above observations indicate that tRNP (Ser)Sec is the most likely target of anti-SLA. List of Abbreviations AIH, autoimmune hepatitis; SLA, soluble liver antigen; tRNP (Ser)Sec , tRNA-associated antigenic protein Competing Interests The author(s) declare that they have no competing interests. Authors' Contributions DPB designed the study, performed the ELISA and inhibition experiments and contributed in writing the report. DG performed the immunoblot testing of anti-enolase antibodies. IB, SL & YM performed the immunoblot experiments involving liver preparations. RRM helped with the artwork. GMV provided clinical material and contributed in writing the report. DV supervised the study and wrote the report. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546408.xml |
521487 | Development of a perioperative medicine research agenda: a cross sectional survey | Background Post-operative complications are a significant source of morbidity and mortality for patients undergoing surgery. However, there is little research in the emerging field of perioperative medicine beyond cardiac risk stratification. We sought to determine the research priorities for perioperative medicine using a cross sectional survey of Canadian and American general internists. Methods Surveys were electronically sent to 312 general internists from the Canadian Society of Internal Medicine and 130 internists from the perioperative medicine research interest group within the US based Society of General Internal Medicine. The questionnaire contained thirty research questions and respondents were asked to rate the priority of these questions for future study. Results The research topics with the highest ratings included: the need for tight control of diabetes mellitus postoperatively and the value of starting aspirin on patients at increased risk for postoperative cardiac events. Research questions evaluating the efficacy and safety of perioperative interventions had higher ratings than questions relating to the prediction of postoperative risk. Questions relating to the yield of preoperative diagnostic tests had the lowest ratings (p < 0.001 for differences across these categories). Conclusion The results of this survey suggest that practicing general internists believe that interventions studies are a priority within perioperative medicine. These findings should help prioritize research in this emerging field. | Background Over 40 million people undergo non-cardiac surgery in the United States each year [ 1 ]. Postoperative cardiac complications affect 2–18% of patients alone, costing over 20 billion dollars annually in the United States [ 2 ]. Many will suffer other potentially avoidable perioperative complications such as pneumonia, hemorrhage or infection. Efforts to minimize these complications have resulted in the development of the field of perioperative medicine. Until now, research in this emerging field has focused primarily on cardiac risk stratification [ 3 ]. Despite the significant medical and economic burden of perioperative complications, few studies evaluate diagnostic testing, risk stratification for non-cardiac complications, or interventions to prevent cardiac or non-cardiac complications. Thus, there is a growing need to expand the research base in this field. Given the wide spectrum of comorbidities in the surgical patient and the potential for postoperative complications, numerous research questions still need to be answered. The purpose of this study is to identify top perceived research priorities in this field using a survey of general internists practicing perioperative medicine. Methods Participants To obtain the opinions of general internists who practice perioperative medicine, from the Canada and the United States, all general internists within the Canadian Society of Internal Medicine (n = 312) and all members of the perioperative medicine interest group of the American based Society of General Internal Medicine (n = 130), were surveyed. Physicians were excluded if they practiced in a subspecialty rather than general medicine (>90% of perioperative medicine consults are performed by general internists [ 4 ]) or did not perform preoperative consultation. Survey development and administration Research questions for the survey were generated from a Medline search of perioperative medicine studies and from a focus group of five general internists active in perioperative medicine research. The questionnaire was pre-tested by four independent general internists for clarity and to confirm face validity. Modifications were made based on this pre-testing. The questionnaire was developed in English and then translated into French for French speaking Canadian physicians by a medical translator. Subsequently, a bilingual general internist validated the French translation. The questionnaire contained 30 randomly ordered research questions conceptually divided into three themes: 1. evaluating the yield of preoperative diagnostic tests (5 questions), 2. predicting postoperative risk (6 questions) and 3. determining the efficacy of perioperative interventions (19 questions). A subset of the sample (130) was asked to specifically rank the importance of these three categories as an internal check of the reliability of the survey instrument. This subset was also asked to list any other research questions that ought to be considered for future research apart from those included in the questionnaire. Respondents were asked to rate the priority for each research question to be studied in the future on a 10 point Likert scale where 1 indicates a low priority study question and 10 is a high priority research question. For questions that have been partially answered by existing studies, respondents were specifically asked to rate the priority of "further research" in these areas. The self- administered questionnaire was electronically mailed in October 2000 (faxes were sent to those without e-mail addresses). For non-responders, second and third mail-outs were sent in November and December 2000. Statistical analyses Descriptive statistics were used to analyze the demographic data and ratings for individual questions. T-tests (two- sided) were used to compare mean response scores across different subgroups. Because of the multiple planned comparisons, the alpha was set at 0.005 to determine statistical significance. Repeated measures analysis of variance was used to analyze differences across the 3 major themes of questions using SPSS statistical software. Results After 3 mailings, we obtained 152 completed surveys (overall response rate 34%). Thirty-three respondents were then excluded because of subspecialty status or because they did not perform perioperative consults. Respondents belonging to the Society of General Internal Medicine special interest group and Canadian Society of Internal Medicine were identical in most respects except fewer Canadian Society of Internal Medicine members had academic appointments (54% vs. 100%). Table 1 shows the demographic and professional characteristics for all respondents. Table 1 Physician characteristics (n = 119) Characteristics Age (SD) 44 (9.2) Female (%) 24 Average year of graduation 1982 Number of years in practice, mean (range) 13 (0.5–35) Practice Location (%) Rural 8 Urban <50 000 8 Urban 50–250 000 25 Urban >250 000 59 Academic appointment (%) 77 Number of preoperative consults performed per month, median (range) 10 (1–100) Research questions evaluating the efficacy of perioperative interventions had higher ratings followed by questions relating to prediction of postoperative risk. Questions evaluating the yield of preoperative diagnostic tests had the lowest ratings. The differences in ratings across these general categories were statistically significant (p < 0.001) and this pattern persisted regardless of academic status or volume of consults seen. Mean scores for individual research questions, based on responses where 1 indicates low priority for future research and 10 indicates high priority, ranged from a low of 3.6 (± 2.3 standard deviation) to a high of 7.2 (± 2.1 standard deviation). Mean scores for the ten highest rated individual questions are given in Table 2 . Only one respondent suggested additional research topics that were not included in the questionnaire. The full list of 30 research topics presented to respondents in the questionnaire is presented in the appendix [see Additional file 1 ]. Table 2 The ten highest rated perioperative research issues: mean scores* (rank) Research issues Total 95% (CI) High** volume Low volume Academic Non-academic The value of tight control of diabetes mellitus postoperatively. 7.2 (1) 6.8–7.6 7.2 (1) 7.1 (2) 7.1 (3) 7.2 (1) Starting aspirin on patients at increased risk for postoperative cardiac complications. 7.1 (2) 6.7–7.5 7.2 (2) 7.0 (4) 7.1 (4) 7.1 (2) Safety and efficacy of continuing aspirin preoperatively for those already taking aspirin. 7.0 (3) 6.6–7.4 7.0 (5) 7.1 (1) 7.1 (5) 7.0 (3) Optimal management of perioperative anticoagulation for patients with prosthetic valves. 7.0 (4) 6.5–7.4 6.9 (6) 7.1 (3) 7.0 (6) 7.0 (4) The value of starting angiotensin converting enzyme inhibitors for those at increased risk of postoperative cardiac complications. 6.9 (5) 6.5–7.3 7.2 (3) 6.7 (9) 7.0 (7) 6.9 (7) Determining the diagnostic yield of routine postoperative cardiac surveillance. 6.8 (6) 6.4–7.2 7.1 (4) 6.6 (13) 6.5 (12) 6.9 (6) Developing interventions to minimize postoperative delirium. 6.8 (7) 6.4–7.2 6.5 (9) 7.0 (6) 6.4 (14) 6.9 (5) The value of starting beta-blockers for patients at increased risk of postoperative cardiac complications. 6.7 (8) 6.2–7.2 6.4 (11) 7.0 (5) 7.2 (1) 6.6 (10) Optimal management of perioperative anticoagulation for patients with atrial fibrillation. 6.6 (9) 6.2–7.1 6.3 (12) 6.9 (7) 6.6 (11) 6.7 (9) Developing a risk stratification index for predicting postoperative pulmonary complications 6.6 (10) 6.2–7.1 6.6 (8) 6.7 (10) 6.5 (13) 6.7 (8) * Mean scores were derived from responses based on a 10-point Likert scale. A score of 10 indicates high priority and 1 indicates low priority for future research. ** Ratings from consultants who see a high volume of preoperative consults (>10/month). Mean scores for most questions were similar among physicians independent of their academic status or whether they performed a high volume of preoperative consults (defined as greater than 10 consults per month) or not. For several questions, however, differences in ranking according to academic status and volume of consultations were found, although none achieved statistical significance (Table 2 ). Discussion There are few areas within perioperative medicine that are well studied beyond the area of predicting cardiac risk. The American Heart Association perioperative guidelines highlighted the paucity of studies on interventions to prevent postoperative cardiac events [ 3 ]. Reflecting this statement, respondents globally rated studies that determine the efficacy and safety of interventions as higher priority for future research compared to studies predicting postoperative risk or determining diagnostic yield of tests. Within the category of intervention studies, questions on medical therapy to prevent postoperative cardiac complications were among the highest rated questions. This result is congruent to the significant prevalence, morbidity and mortality associated with postoperative cardiac complications. A study by Devereaux et al also identified this area as an important target for future research after finding considerable practice variation in the management of cardiac medications [ 4 ]. Innovative interventions for cardiac protection with antiplatelet agents, angiotensin converting enzyme inhibitors or tight glycemic control were highly rated. Despite the publication of small trials of beta blocker therapy, responding internists felt that there was a need to definitively determine the efficacy of perioperative beta blockade. Respondents may be more skeptical of adopting results from the small trials of beta blocker where methodological controversies have arisen [ 5 ]. Other intervention questions that were rated highly focused on understudied areas of perioperative anticoagulation. Determining optimal perioperative anticoagulation strategies for patients with prosthetic valves or atrial fibrillation was rated highly. Although perioperative risk stratification is well studied for identifying those at risk of cardiac events, pulmonary complications occur more frequent than cardiac complications and are associated with a longer hospital stay [ 6 ]. Reflecting this significant morbidity and cost of respiratory complications, development of a prediction rule for postoperative pulmonary complications was among the highest rated topics. Since the completion of the survey, the ratings also reflect ongoing research activity within perioperative medicine. Tight glycemic control was a high rated topic and a study examining the effect of tight glycemic control in postoperative patients in a critical care setting has been published since the completion of this survey. Also since the completion of this survey, a large multi-center trial examining the efficacy of perioperative beta blockade has been launched. Another highly rated research topic was developing a prediction rule for pulmonary complications. Recently, a study was published to identify those at increased risk of postoperative pneumonia. Two of the highly rated research priority topics have been recently published in major medical journals [ 7 , 8 ]. There are several potential limitations with this study. The low response rate may be felt to limit the generalizability to other general internists. However, views of those who practice perioperative medicine and who have a particular interest in the area, the research consumers, are important in developing a research agenda. We presume that physicians who practice within an area and who have a particular interest are more likely to respond to a survey than those who do not. Thus, the physicians who responded to the questionnaire and reported practicing perioperative medicine define the group of research consumers we were targeting. This study also only examined the beliefs of general internists on research priorities since greater than 90% of the perioperative medicine consultations are conducted by general internists rather than subspecialists in internal medicine in tertiary care centers [ 4 ]. Also, lower response rates are seen in physician surveys but surveys with response rates of 10–45% are published in major medical journals [ 9 - 15 ]. Another limitation is that anesthesiologists, cardiologists or primary care physicians perform perioperative medicine consultations and their opinions were not elicited in this survey. Another potential limitation is that not all important research questions could be examined. However, only one respondent indicated additional research topics suggesting that there were no major omissions in the research topics listed. Additionally, it is difficult to interpret meaningful differences in mean Likert response scores among individual questions. Individual scores and their confidence intervals should be interpreted to give a general idea of high- priority questions, rather than a strict ranking of research pursuits. Conclusion Perioperative medicine research is growing in response to the significant medical and economic consequences of perioperative complications. Identifying the research priorities of those who provide perioperative medical care – the consumers of research, is important. The results suggest that intervention studies are a higher priority for future research compared to studies that predict postoperative complications or determine the yield of diagnostic tests. Researchers and funding boards may use the findings of this survey to identify perceived high priority research topics within perioperative medicine. Competing interests None declared. Authors' contribution NK, TT, AF, NC, FAM, and WG contributed to the design of the project. NK and TT contributed to data collection and data analysis. NK, TT, WG contributed to writing the manuscript. AF, NC, FAM made substantive, intellectual editing contributions. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Perioperative medicine research questions. This appendix lists all 30 perioperative research issues from the survey, along with the mean scores rated by all respondents. Click here for file | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521487.xml |
551606 | Evaluation of an education and activation programme to prevent chronic shoulder complaints: design of an RCT [ISRCTN71777817] | Background About half of all newly presented episodes of shoulder complaints (SC) in general practice are reported to last for at least six months. Early interventions aimed at the psychological and social determinants of SC are not common in general practice, although such interventions might prevent the development of chronic SC. The Education and Activation Programme (EAP) consists of an educational part and a time-contingent activation part. The aim of the EAP is to provide patients with the proper cognitions by means of education, and to stimulate adequate behaviour through advice on activities of daily living. Design The article describes the design of a randomised clinical trial (RCT) to evaluate the effectiveness and cost-effectiveness of an EAP in addition to usual care, compared to usual care only, in the prevention of chronic SC after six months. It also describes the analysis of the cost and effect balance. Patients suffering from SC for less than three months are recruited in general practice and through open recruitment. A trained general practitioner or a trained therapist administers the EAP. Primary outcome measures are patient-perceived recovery, measured by self-assessment on a seven-point scale, and functional limitations in activities of daily living. Questionnaires are used to study baseline measures, prognostic measures, process measures and outcome measures. Discussion The inclusion of patients in the study lasted until December 31 st 2003. Data collection is to end in June 2004. | Background Shoulder complaints Shoulder complaints (SC) have been defined by Sobel & Winters [ 1 ] as pain localised in the region of the deltoid muscle, the acromioclavicular joint, the superior part of the trapezoid muscle and the scapula. Radiation of the pain to the arm as well as limitation of the motion of the upper arm and/or the shoulder girdle may be present [ 1 ]. SC are characterised by pain in the area between the base of the neck and the elbow, at rest or when elicited by movement of the upper arm (Fig. 1 ). Figure 1 Area between the base of the neck and the elbow Musculoskeletal disorders, of which SC constitute the second largest group after low back disorders, account for the second largest share in healthcare costs and represent the largest group of work-related diseases in the Netherlands [ 2 ]. Shoulder complaints in general practice The point prevalence of SC in the general population in the Netherlands has recently been estimated at 21% [ 3 ]. In a British study a lower point prevalence of 14% has been found [ 4 ]. The annual incidence of SC as seen by general practitioners (GPs) in the Netherlands lies between 15 and 25 patients per 1000 registered general practice patients[ 1 ]. About half of all newly presented episodes in general practice are reported to last for at least six months, while 40 percent of the newly presented episodes result in disability in terms of activities of daily living after one year [ 5 ]. The International Association for the Study of Pain (IASP) regards persistent or recurring pain lasting less than three months as acute pain, whereas more than three months of persistent or recurring pain is considered to be chronic pain [ 6 ]. The study by van der Windt [ 5 ] showed that, according to this cut-off criterion, 51% of patients with a newly presented episode of SC in general practice develop chronic SC, that is, complaints lasting more than three months. The Dutch College of General Practitioners provides clinical guidelines for the treatment of SC [ 7 ]. These guidelines, however, do not include treatment aimed at psychosocial factors such as maladaptive behaviour and inadequate cognitions, known to play a role in the development and persistence of chronic musculoskeletal diseases [ 8 - 10 ]. Treatments addressing such factors are mentioned in the guidelines, but only as a last resort, when the biomedical approach has proved ineffective in reducing the pain. To date, early interventions aimed at the psychological and social determinants of SC are not common in general practice, although such interventions in the early stages of the SC might prevent the development of chronic complaints [ 11 ]. Early intervention in general practice We hypothesised that an intervention concentrating on psychological and social determinants in the early stages of SC would prevent the development of inadequate cognitions and maladaptive behaviour, ensuring that such inadequate cognitions and behaviour do not play a role in the development of chronic SC later on. The term cognitions refers to the way patients think about their pain and what the pain means to them, in terms of thoughts, beliefs, attitudes and self-efficacy expectations [ 12 ], whereas behaviour refers to the patients' observable actions [ 13 ]. Patients' cognitions and behaviour should thus be influenced so as to become adequate cognitions and adaptive behaviour. A relatively brief treatment in the early stages of SC, administered by a trained therapist, may be expected to be effective in preventing the development of chronic SC. The Education and Activation Programme (EAP) that formed the subject of the present study is such an early intervention. Aim of the study This paper describes the design of a study to evaluate the clinical effectiveness of an early EAP aimed at using psychological and behavioural factors to prevent chronic SC. In addition, the study is to evaluate the balance between costs and effects. EAP in patients with acute SC is to be compared with treatment according to the Dutch College of General Practitioners guidelines. The Medical Ethics Committee of the Institute for Rehabilitations Research in association with Rehabilitation Foundation Limburg has approved the design of the study presented here. Funding was obtained from the Netherlands Organisation for Scientific Research. This paper describes also the rationale and content of the EAP. Education and activation programme Previous studies have indicated that cognitive behavioural therapies aimed at bio-psychosocial factors are promising instruments for the prevention of chronic musculoskeletal pain [ 14 ]. The EAP focuses on the same elements as cognitive behavioural therapies, but is applied at an earlier stage than such therapies [ 15 ]. Whereas the latter focus mainly on the elimination of inadequate cognitions and maladaptive behaviour after they have already developed in the course of the SC, the EAP focuses on guiding the patient towards adequate pain behaviour and reinforcing this behaviour at an early stage of the SC. The aim of the EAP is to prevent the development of inadequate cognitions and maladaptive behaviour in patients with acute SC. Education is used to maintain or induce adequate cognitions by providing information that is tailored to questions that patients have about their SC. Health care educators frequently assume that giving information equals comprehension, which should automatically translate into changed behaviours as the knowledge is applied [ 16 ]. According to Hussey, however, simply receiving a message hardly correlates with understanding it [ 17 ]. Effective learning also requires the active participation of the patient[ 18 ]. Since inadequate cognitions and maladaptive behaviours are not yet fully developed in patients with acute SC, the focus of the EAP is not on restructuring inadequate cognitions or modifying maladaptive behaviour, but on maintaining or inducing the proper cognitions by education and on maintaining or inducing adequate behaviour by giving advice on activities of daily living. Adequate behaviour is considered to be behaviour in which the patient remains active. A comprehensive description of the EAP is given in the Design section. Design Patients Patients are recruited by GPs and in the open population by advertising in local newspapers. Patients are eligible for inclusion in the Randomised Clinical Trial (RCT) if they consult their own GP or respond to adverts in a local newspaper with a new episode of SC that has lasted no longer than three months, at rest or when elicited by movement in the shoulder area. Patients are included if they are 18 years or older and living in the south of the Netherlands. Only newly presented episodes of SC are considered, that is, patients who have not consulted their GP and have not been treated for their SC in the preceding three months. Additional exclusion criteria are given in table 1 . Table 1 Exclusion criteria • other episodes of SC in the 12 months preceding the consultation with the GP • prior fractures and/or surgery of the shoulder • (suspected) referred pain from internal organs • SC with a confirmed extrinsic cause • inability to complete a questionnaire independently • presence of dementia or other severe psychiatric abnormalities Randomised Clinical Trial A Randomised Clinical Trial (RCT) with a six-month follow-up is used to evaluate the effectiveness and cost-effectiveness of an EAP to prevent chronicity in patients with acute SC, compared to usual care. A computer-generated random sequence table is used to randomise the patients to EAP or usual care. Neither the patient nor the GP, nor the trained therapist, can be blinded for the allocated treatment. The trained therapist is also the researcher coordinating the RCT and conducting the data analysis, but is blinded for treatment allocation during the data analysis. The allocation code will be revealed only after the data analysis has been completed. Treatments Usual care (UC) is applied according to the Dutch College of General Practitioners guidelines for SC (version 1999)[ 19 ]. Management during the first two weeks consists of a wait-and-see policy with information and advice about shoulder complaints, possibly supplemented with analgesics or nonsteroidal anti-inflammatory drugs. If this approach has little or no effect, up to three corticosteroid injections can be given. Physiotherapy is considered for complaints persisting after six weeks or more. If the SC persist, referral to a hospital-based specialist may be considered. The focus of the EAP is to maintain or induce the proper cognitions by education and to stimulate adequate behaviour by means of advice on activities of daily living. Table 2 shows the components of the EAP. The EAP is administered by specially trained GPs or an ambulant therapist (CDB) trained to provide the EAP. The ambulant therapist administers the EAP when no trained GP is available in the living area of the patient. Table 2 Elements of the education and activation programme Education • Information on the origin, nature and prognosis of the SC • Information on possible interventions and their effects (tailored to the patient's questions and needs) • Information on the effect of cognitions and behaviour on the perpetuation of the SC Activation When no alterations in activities have occurred due to the SC • Positive reinforcement • Instruction to be aware of possible changes When alterations in activities have occurred due to the SC • Identification of up to three altered frequent activities of daily living • Determination of the desired level of activity and the size of the steps needed to reach this level The EAP consists of a minimum of two sessions and a maximum of six follow-up sessions over a period of six weeks. Each session may last up to 20 minutes. The first and second sessions are organised in the general practice setting by the trained GP, or at the patient's home by the ambulant EAP therapist. The other sessions are provided by telephone. Education The first part of the EAP has an educational purpose, and focuses on information about the origin, nature and prognosis of the SC, possible interventions and their effects, the impact on activities of daily living and its consequences and the patient's own possibility to contribute to recovery. This information is tailored to the patients' questions and needs and is based on the information available in the Dutch College of General Practitioners guidelines for SC. In addition, the effect of cognitions and behaviour on the perpetuation of the SC is clarified to the patient by an example. If possible, this example refers to a condition or circumstance the patient has experienced, such as a broken bone or back pain. The patient is helped by the trained GP or the trained therapist to explore whether his or her thoughts about the SC are justified. Negative patterns of thinking are modified into adequate and accurate thoughts. Activation The second part of the EAP consists of a time contingent activation programme, based on the principles of operant learning. It focuses on gradually increasing activities of daily living, despite the pain. Potential avoidance of activities is countered by reinforcement of continuation or resumption of usual activities. Positive reinforcement is used to stimulate patients with a normal activity pattern, in spite of their SC, to continue their activities. This positive reinforcement may be enough to achieve continuation of the desired activities [ 13 ]. These patients are also instructed to be aware of possible changes in their activities that could lead to undesirable behaviour such as reduced use of the affected shoulder. Patients who have reduced their normal activities are helped to identify up to three frequent activities of daily living that they have reduced as a result of the SC. These activities are stepwise gradually increased to the desired level of activity in a time-contingent manner. The desired level of activity and the magnitude of the increases are determined and agreed upon by the EAP therapist and the patient. The patient and the EAP therapist also plan a progress evaluation, which is used to positively reinforce the patient's behaviour if the gradual increase has been correctly implemented or to adjust the magnitude of the increases if the original objectives prove too optimistic. Measurements The first outcome measure is the perceived recovery of the patient. Patients are considered to be recovered when they report to be much improved or fully recovered, on an 7-point ordinal scale, after six months. The second outcome measure is that of functional limitations in activities of daily living. This variable is assessed by a 16-item questionnaire, the shoulder disability questionnaire (SDQ)[ 20 ], with a scoring range of 0 to 16. A reduction of the score on this questionnaire implies a reduction in functional limitations. The outcome measures are recorded at 6, 12 and 26 weeks after randomisation. The SDQ is also measured at baseline. A cost diary [ 21 ] is used to assess health care utilisation, direct non-medical costs and indirect costs. A complete overview of baseline measures, prognostic measures, process measures and outcome measures is given in table 3 . Table 3 Variables Baseline measures T = 0 Demographic variables • Age • Gender • Employment status Specific disease characteristics • Affected side • Possible cause of shoulder complaints • Duration of complaints • History of shoulder complaints Co-morbidity Physical activity Workload Treatment credibility and preference Prognostic measures T = 0 Mobility of glenohumeral joint • HIB (hand in back), HIN (hand in neck), passive exorotation • Active and passive abduction Mobility of cervicothoracal spine Severity of main complaint Psychosocial variables • Anxiety 1 • Depression 1 • Somatisation 1 • Distress 1 Job content Outcome measures T = 1,2,3 Perceived recovery of complaints Functional limitations to daily activities 2 Process measures T = 0,1,2,3 Psychosocial variables • Kinesiophobia 3 • Fear avoidance and beliefs 4 • Catastrophising 5 • Coping with pain 5 • Internal locus of control 5 • External locus of control 5 Global assessment Shoulder pain 6 General health 7 Cost T = 0–26 weeks Health care utilisation 8 Direct non-medical costs Indirect costs 1 four-dimensional complaint list [25] 2 Shoulder Disability Questionnaire [20] 3 Tampa Scale for Kinesiophobia – Dutch version (partly) [26] 4 Fear Avoidance and Beliefs Questionnaire – Dutch version (partly) [27] 5 Pain Coping and Cognition List [28] 6 Shoulder Pain Score [29] 7 Generic Health Related Quality of Life [30] 8 Cost Diary [21] Data analysis The statistical analysis will be carried out according to the 'intention-to-treat' principle. Differences between groups, with 95% confidence intervals, will be calculated for each outcome measure. The study groups will be compared by an independent samples t-test for changes since baseline for continuous outcome variables and the chi-square test for categorical outcome variables. In addition, the corresponding baseline value for each continuous outcome will be used as a covariate. The analysis will be repeated taking any loss-to-follow-up into account by applying a sensitivity analysis in which all patients who are lost to follow-up are first considered to show the largest observed improvement and then the largest observed deterioration in outcome measures. The analyses of the difference in change for the outcomes at three and six months will account for the repeated measures character of the data. Baseline characteristics that are a priori considered to be possible prognostic factors for outcome variables, as well as post-randomisation differences between the groups, will be handled as potential confounders. Their influence will be evaluated by means of multivariable regression analyses. In the case of confounding, adjusted effect estimates will also be reported. Sample size About half of all newly presented episodes of SC in general practice are reported to last for at least six months. A number needed to treat of 4.5 after six months is considered clinically relevant. This implies an absolute reduction of 22% of the proportion of patients with SC after six months. With a two-sided alpha of 0.05 and a statistical power (1-β) of 0.80, 70 patients per treatment group are needed to detect a difference in favour of the EAP compared to usual care after six months. Embedding in the Dutch Shoulder Disability Study This RCT is part of the Dutch Shoulder Disability Study, a comprehensive prognostic cohort study on SC, with randomised controlled interventions in subcohorts. The Dutch Shoulder Disability Study is funded by the Netherlands Organisation for Scientific Research (NWO, grant number 904-65-901). Discussion Reasons for publishing a study design There are several reasons to publish a study design before the results are available. The main reason is that it provides an opportunity to counteract publication bias, that is, the phenomenon whereby a study producing positive results is more likely to be published than a study showing no difference between the study groups [ 22 ], [23]. Hence, if the design is published but not the results, the study can still be included in a systematic review because data can be retrieved from the researcher [24]. Another reason is that it gives researchers the opportunity to reflect upon the study design independently of the results. When results run counter to the researchers' expectations, methodological flaws are usually examined. But when the results are in line with expectations, methodological flaws are more likely to be overlooked. [ 22 ] The third reason arises from the tendency among randomised controlled studies to deviate from their original designs, mainly because of practical problems. Such deviations from the study design may affect the study results. Publishing the study design forces researchers to test its implementation and to answer for any deviations from the design. Finally, this article offers us an opportunity to describe the rationale and content of the intervention in greater detail than the methods section of an article reporting the results of the RCT would do [24]. Applicability in general practice The EAP is a brief intervention that can easily be administered by GPs in addition to the usual care according to the guidelines. This might give GPs an instrument to prevent the development of chronic SC in the early stages of the complaints by focusing on psychological and social determinants. Time schedule The inclusion of patients in the study lasted until December 31 st 2003. Data collection will be completed in June 2004. Currently, 108 patients have been included and are being followed up. Abbreviations SC: Shoulder Complaints GP: General Practitioner IASP: International Association for the Study of Pain EAP: Education and Activation Programme RCT: Randomised Clinical Trial UC: Usual Care CDB: Camiel De Bruijn SDQ: Shoulder Disability Questionnaire NWO: Netherlands Organisation for Scientific Research Competing interests The author(s) declare that they have no competing interests. Authors' contributions CDB participated in the design of the study, will provide the EAP as an ambulant therapist, coordinate the data collection, will perform the statistical analysis and publish the results. RDB participated in the design of the study and will participate in the statistical analysis. JG, MG, WVDH and G-JD also participated in the design of the study. AK participated in the development of the education and activation programme and will conduct the training of the general practitioners that will give the education and activation programme. GVDH conceived of the study, and participated in its design. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551606.xml |
544578 | Nanoparticles – known and unknown health risks | Manmade nanoparticles range from the well-established multi-ton production of carbon black and fumed silica for applications in plastic fillers and car tyres to microgram quantities of fluorescent quantum dots used as markers in biological imaging. As nano-sciences are experiencing massive investment worldwide, there will be a further rise in consumer products relying on nanotechnology. While benefits of nanotechnology are widely publicised, the discussion of the potential effects of their widespread use in the consumer and industrial products are just beginning to emerge. This review provides comprehensive analysis of data available on health effects of nanomaterials. | 1. Introduction Scientists world-wide are continuing to discover unique properties of everyday materials at the sub micrometer scale [ 1 , 2 ]. This size domain is better known as nano- (a billionth) meter domain. These novel properties of common materials observable only at the nano-scale dimensions have already found their first commercial applications [ 3 ]. For example, nanomaterials are present in some sunscreens, toothpastes, sanitary ware coatings and even food products. Manmade nanoparticles ranges from the well-established multi-ton production of carbon black and fumed silica for applications in plastic fillers and car tyres to microgram quantities of fluorescent quantum dots used as markers in biological imaging. As nano-sciences are experiencing massive investment worldwide [ 4 , 5 ], there will be a further rise in consumer products relying on nanotechnology [ 6 ]. While benefits of nanotechnology are widely publicised, the discussion of the potential effects of their widespread use in the consumer and industrial products are just beginning to emerge [ 7 , 8 ]. Both pioneers of nanotechnology [ 9 ] and its opponents [ 10 ] are finding it extremely hard to argue their case as there is limited information available to support one side or the other. It has been shown that nanomaterials can enter the human body through several ports. Accidental or involuntary contact during production or use is most likely to happen via the lungs from where a rapid translocation through the blood stream is possible to other vital organs [ 11 ]. On the cellular level an ability to act as a gene vector has been demonstrated for nanoparticles [ 12 ]. Carbon black nanoparticles have been implicated in interfering with cell signalling [ 13 ]. There is work that demonstrates uses of DNA for the size separation of carbon nanotubes [ 14 ]. The DNA strand just wraps around it if the tube diameter is right. While excellent for the separation purposes it raises some concerns over the consequences of carbon nanotubes entering the human body. In this review we summarise the known facts about nanomaterial hazards, discuss the potential entry points of nanoparticles into the human body, explore their likely pathways inside the body and analyse published experimental results on the bioactivity of nanomaterials. 2. General background Human skin, intestinal tract and lungs are always in direct contact with the environment. Whereas skin acts as a barrier, lungs and intestinal tract also allow transport (passive and/or active) of various substances like water, nutrients or oxygen. Because of that fact they are likely to be a first port of entry for nanomaterials journey into the human body. Our knowledge in this field mainly comes from drug delivery (pharmaceutical research) and toxicology (xenobiotics) studies. Human skin functions as a strict barrier and no essential elements are taken up through the skin (except radiation necessary to build up vitamin D). The lungs exchange oxygen and carbon dioxide with the environment, and some water escapes with warm exhaled air. The intestinal tract is in close contact with all the materials taken up orally; there all nutrients (except gasses) are exchanged between the body and the environment. The histology of the environmental contact sides of these three organs is significantly different. The skin of an adult human is roughly 1.5 m 2 in area, and is at most places covered with a relatively thick first barrier (10 micron) which is build of strongly keratinised dead cells (Fig 1 ). This first barrier is difficult to pass for ionic compounds as well as water soluble molecules. Figure 1 schematic representation of human skin; Stratum corneum is the top of the five layers making epidermis, it is composed of keratinised dead cells glued by lipids. It is shed off and replaced every two weeks. Depending on the part of the body its thickness varies from 0.05 mm to 1.5 mm. The lung consists of two different parts, airways (transporting the air in and out the lungs) and alveoli (gas exchange areas). Human lungs contain about 2300 km of airways and 300 million alveoli (gas exchange areas) (Fig 2 ). The surface area of the lungs is 140 m 2 in adults, as big as a tennis court. The airways are a relatively robust barrier, an active epithelium protected with a viscous layer of mucus. In the gas exchange area, the barrier between the alveolar wall and the capillaries is very thin. The air in the lumen of the alveoli is just 0.5 micron away from the blood flow. The large surface area of the alveoli and the intense air-blood contact in this region makes the alveoli less well protected against environmental damage when compared with airways. Figure 2 Cross-section of alveoli; Schematic cross-section of alveoli showing a very thin (500 nm) separation between blood and air. An SEM image of the alveoli is shown in the inset. The intestinal tract is a more complex barrier – exchange side, it is the most important portal for macromolecules to enter the body. From the stomach, only small molecules can diffuse through the epithelium. The epithelium of the small and large intestines is in close contact with ingested material so that nutrients can be utilized. A mixture of disaccharides, peptides, fatty acids, and monoglycerides generated by digestion in small intestine are further transformed and taken in the villi (Fig 3 ). Villi, in turn, are covered with micro-villi, which bring overall surface available to nutrients to 200 square meters. Figure 3 Villi in small intestine; A surface structure of villi covered with micro-villi is dramatically multiplies the area of gastero-intestine tract to 200 m 2 . Inset shows an SEM image of villi. 3. Lung 3.1 Inhalation and pulmonary clearing of insoluble solids The pathogenic effects of inhaled solid material depend primarily on achieving a sufficient lung burden [ 15 ]. The lung burden is determined by the rates of deposition and clearance. Logically, for any dust or fibre, a steady-state dose level will be achieved when the rates come into balance. This is only true when the solid material does not interfere with the clearance mechanisms. In respect to the burden the chemical and physical properties of the material itself are important insofar as they influence deposition and clearance rates. Spherical solid material can be inhaled when its aerodynamic diameter is less than 10 micron. The smaller the particulates the deeper they can travel into the lung, particles smaller than 2.5 micron will even reach the alveoli. Ultrafine particles (nanoparticles with an aerodynamic diameter of less than 100 nm) are deposited mainly in the alveolar region. Fibres are defined as solid materials with a length to diameter ratio of at least 3:1. Their penetration into the lung depends on the aerodynamic properties. Fibres with a small diameter will penetrate deeper into the lungs, while very long fibres (>>20 micron) are predominantly stuck in the higher airways [ 16 - 21 ]. The mucociliary escalator dominates the clearance from the upper airways; clearance from the deep lung (alveoli) is predominantly by macrophage phagocytosis. The mucociliary escalator is an efficient transport system pushing the mucus, which covers the airways, together with the trapped solid materials towards the mouth. The phagocytosis of particles and fibres results in activation of macrophages and induces the release of chemokines, cytokines, reactive oxygen species, and other mediators; this can result in sustained inflammation and eventually fibrotic changes. The phagocytosis efficiency can be affected by the (physical-chemical) characteristics of the solid material (see below); moreover, fibres too long to be phagocytized (fibres longer than the diameter of the alveolar macrophage) will only be cleared very slowly. Laboratory exposure studies have shown that if the inhaled concentrations are low, such that the deposition rate of the inhaled particles is less than the mechanical alveolar macrophage-mediated clearance rate in the lung, then the retention half time is about 70 days (steady-state lung burden during continuous exposure). If the deposition rate of the inhaled particles exceeds this clearance rate, the retention half time is significantly increased, reflecting an impaired or prolonged alveolar macrophage-mediated clearance function with continued accumulation of lung burden (overload). Inhaled fibres, which are persistent in the alveoli, can interact with the pulmonary epithelial cells or even penetrate the alveolar wall and enter the lung tissue. These fibres are often described as being in the "interstitial" where they may lie between or within the cells making up the alveolar walls. Bio-persistent solid materials, certainly those particles containing mutagenic substances or asbestos fibres or silica, which remain for years in the lungs, increase the risk of developing cancer. 3.2 Deposition and clearing of solid nanomaterials It has been reported recently that nanotubes show a sign of toxicity [ 22 ], confirmed in two independent publications by Warheit et al [ 23 ] and Lam et al [ 24 ], which demonstrated the pulmonary effects of single walled cabon nanotubes in vivo after intratracheal instillation, in both rats and mice. Both groups reported granuloma formation, and some interstitial inflammation. The research group of Warheit et al [ 23 ] concluded that these findings (multifocal granulomas) may not have physiological relevance, and may be related to the instillation of a bolus of agglomerated nanotubes. But for the authors of [ 24 ] their results indicate that if carbon nanotubes reach the lungs, they are much more toxic than carbon black and can be more toxic than quartz. These studies have to be read with some caution because a study by the National Institute for Occupational Safety and Health (NIOSH) showed that none or only a small fraction of the nanotubes present in the air can be inhaled [ 25 ]. Clearance from the lung depends not only on the total mass of particles inhaled but also on the particle size and, by implication, on particle surface, as shown in the following studies. A sub-chronic 3 months inhalation exposure of rats to ultrafine (~20 nm) and fine (~200 nm) titanium dioxide (TiO2) particles demonstrated that the ultrafine particles cleared significantly slower, showed more translocation to interstitial sites and to regional lymph nodes when compared to the fine TiO2 particles [ 26 ]. By comparing carbon black particles of similar size and composition but with significant specific surface area difference (300 versus 37 m 2 /g), it was found that the biological effects (inflammation, genotoxicity, and histology) were dependent on specific surface area and not particle mass. Similar findings were reported in earlier studies on tumorigenic effects of inhaled particles. It was shown that tumour incidence correlated better with specific surface area than with particle mass [ 27 , 28 ]. Comparing the health effects of chronically inhaled TiO2 particles with distinctly different sizes, it is remarkable that the low exposure (10 mg/m 3 ) study [ 29 ] resulted in a greater lung tumour incidence than the high exposure (250 mg/m 3 ) study [ 30 ]. The inhaled particles in both studies consisted of aggregated primary particles, with an aerodynamic diameter that was probably not very different. The primary particle size of the low dose study was 20 nm, while it was approximately 300 nm in the latter study. In summary, most nano-sized spherical solid materials will easily enter the lungs and reach the alveoli. These particles can be cleared from the lungs, as long as the clearance mechanisms are not affected by the particles themselves or any other cause. Nano-sized particles are more likely to hamper the clearance resulting in a higher burden, possibly amplifying any possible chronic effects caused by these particles. It is also important to note that specific particle surface area is probably a better indication for maximum tolerated exposure level than total mass. Inhaled nano-fibres (diameter smaller than 100 nm) also can enter the alveoli and their clearing would, in addition, depend on the length of the specific fibre. Recent publications on the pulmonary effects of carbon nanotubes confirm the intuitive fear that nano-sized fibre can induce a rather general non-specific pulmonary response. 3.3 Particle surface and biocompatibility Reports on the surface properties of nanoparticles, both physical and chemical, stress that nanoparticles differ from bulk materials. Their properties depend heavily on the particle size. Therefore, nanoparticles are not merely small crystals but an intermediate state of matter placed between bulk and molecular material. Independently of the particle size, two parameters play dominant role. The charges carried by the particle in contact with the cell membranes and the chemical reactivity of the particle [ 31 ]. 3.3.1 Surface charges Polycationic macromolecules show a strong interaction with cell membranes in vitro. A good example can be found in the Acramin F textile paint system. Three poly-cationic paint components exhibited considerable cytotoxicity (LD50 generally below 100 mg/ml for an incubation of 20–24 hours) in diverse cell cultures, such as primary cultures of rat and human type II pneumocytes, and alveolar macrophages and human erythrocytes. The authors argued that the multiple positive charges play an important role in the toxic mechanism [ 32 , 33 ]. Biocompatibility studies [ 34 ] revealed that the cytotoxicity of polycationic materials such as DEAE-dextran and poly-L-lysine (PLL) [ 35 , 36 ], dendrimers [ 37 ] and polyethylenimine (PEI) [ 38 ] increases with the increase in their molecular weight. However, these findings apply only to polymers having same chemical structure, but not for different types of polycations. Consequently, to explain the toxicity of polymers with different structures further parameters have to be taken into account. Dekie et al [ 39 ] concluded that the presence of a primary amine group on poly L-glutamic acid derivatives has a significant toxic effect on red blood cells causing them to agglutinate. Not only the type of amino function but also the charge density resulting from the number and special arrangement of the cationic residues is an important factor for cytotoxicity. Ryser [ 40 ] suggested that a three-point attachment is necessary for eliciting a biological response on cell membranes, and argued that the activity of a polymer will decrease when the space between reactive amine groups is increased. The arrangement of cationic charges depends on the three-dimensional structure and flexibility of the macromolecules and determines the accessibility of their charges to the cell surface. For example, branched molecules were found to be more efficient in neutralising the cell surface charge than polymers with linear or globular structure, as rigid molecules have more difficulties to attach to the membranes than flexible molecules [ 41 ]. Therefore, high cationic charge densities and highly flexible polymers should cause higher cytotoxic effects than those with low cationic charge densities. Globular polycationic macromolecules (cationised Human Serum Albumine (cHSA), ethylenediamine-core poly(amidoamine) dendrimers (PAMAM) were found to be polymers with a good biocompatibility (low cytotoxicity), whereas polymers with a more linear or branched and flexible structure (e.g. polydiallyldimethylammonium chloride (DADMAC), PLL, PEI) showed higher cell damaging effects. 3.3.2 The surfactant interaction and surface chemistry Geiser et al [ 42 ] studied the influence of the particle surface chemistry on its interaction with the lung's surface-lining layer. They found that, regardless of the nature of their surfaces, particles will be submersed into the lining layer after their deposition in small airways and alveoli. This displacement is promoted by the surfactant film itself, whose surface tension falls temporarily to relatively low values [ 42 , 43 ]. On the other hand, reactive groups on a particle surface will certainly modify the biological effects. For silica, it has been shown that surface modification of quartz affects its cytotoxicity, inflammogenicity and fibrogenicity. These differences are mainly due to particle surface characteristics [ 44 ]. Specific cytotoxicity of silica is strongly correlated with the appearance of surface radicals and reactive oxygen species (ROS), which is considered to be the key event in the development of fibrosis and lung cancer by this compound [ 45 ]. Although the type of particle does not seem to play an important role in whether it is embedded in the surfactant lining of the alveoli, the embedding process itself is crucial. Particle-cell interaction is possible only after the immersion of the particulates in the lining fluid and research is needed to study this phenomenon in detail in relation to inhaled nanoparticles. Logically, as described in the report for silica [ 45 ], the reactive groups on nanoparticles influence their interaction with the lung (or more general with biological material). In some instances it might be possible to predict the reactivity of the nano-surface. However, considering the scarcity of data, it would be sensible to verify these predictions by some laboratory testing. 3.4 Systemic translocation of inhaled particles The impact of inhaled particles on other organs has only recently been recognised. Most research has concentrated on the possible consequences of particle related malfunction of the cardio-vascular system, such as arrhythmia, coagulation [ 46 ] etc. However, recent data support the concept that the autonomic nervous system may also be a target for the adverse effects of inhaled particulates [ 47 , 48 , 11 ]. Two complementary hypotheses explain the cardiovascular malfunctions after inhalation of ultra-fine particles. The first hypothesis explains the observed effects by the occurrence of strong (and persistent) pulmonary inflammatory reactions in the lungs, leading to the release of mediators (see above), which may influence the heart, coagulation, or other cardiovascular endpoints. The second hypothesis is that the particles translocate from the lungs into the systemic circulation and thus, directly or indirectly, influence haemostasis or cardiovascular integrity. In the evaluation of the health effects of inhaled nanoparticles the translocation to the systemic circulation is an important issue. Conhaim and co-workers [ 49 ] found that the lung epithelial barrier was best fitted by a three-pore-sized model, including a small number (2%) of large-sized pores (400-nm pore radius), an intermediate number (30%) of medium-sized pores (40-nm pore radius), and a very large number (68%) of small-sized pores (1.3-nm pore radius). The exact anatomical location of this structure, however, remains to be established (see the review by Hermans and Bernard [ 50 ]). Until recently, the possible passage of xenobiotic particles has not been attracting much attention, although, the concept is now gaining acceptance in pharmacology for the administration of macromolecular drugs by inhalation [ 51 ]. Nemmar et al [ 11 ] studied the particle-translocation of inhaled ultrafine technetium ( 99m Tc) labelled carbon particles to the blood. These particles, which are very similar to the ultrafine fraction of actual pollutant particles, diffused rapidly – within 5 minutes – into the systemic circulation (Fig 4 ). The authors concluded that phagocytosis by macrophages and/or endocytosis by epithelial and endothelial cells are responsible for particle-translocation to the blood but other roots must also exist. Figure 4 Translocation of inhaled ultrafine particles. Time-activity curve over liver and bladder expressed as percent of initial lung radioactivity. Insert, Whole body gamma camera image of 1 representative volunteer recorded at 60 minutes. The radioactivity over the organs is expressed as counts per minute (CPM) per pixel within each region of interest (ROI). The values recorded over the stomach were not included because this radioactivity may also come partly from swallowing of particles deposited in the mouth. Reproduced with permission from Nemmar et al , "Passage of inhaled particles into the blood circulation in humans", Circulation 2002;105(4):411-41. The literature on the translocation of very small particles from the lungs into the blood circulation is limited and often conflicting. A recent study has reported deposition and clearance over 2 h of an ultrafine (60 nm) 99m Tc labelled aerosol in human volunteers. No significant radioactivity was found in the liver (1–2 % of the inhaled radioactivity) but, unfortunately, no radioactivity measurements with blood were reported [ 52 ]. In agreement with findings of Nemmar et al [ 11 ], Kawakami et al. [ 53 ] have reported the presence of radioactivity in blood immediately after inhalation of 99mTc-technegas in human volunteers. It is also known [ 54 ] that aerosolised insulin gives a rapid therapeutic effect although the pathways for this translocation are still unclear. In addition to human studies, in experimental animal studies, we [ 11 ] and others [ 55 , 16 , 57 ] have reported extra-pulmonary translocation of ultrafine particles after intra-tracheal instillation or inhalation. However, the amount of ultrafine particles that translocate into blood and extra-pulmonary organs differed among these studies. It has also been shown that, following intranasal delivery, polystyrene microparticles (1.1 micron) can translocate to tissues in the systemic compartment [ 58 ]. A recent study [ 59 ] has provided, for the first time, morphological data showing that inhaled polystyrene particles are transported into the pulmonary capillary space, presumably by trans-cytosis. Another alley of translocation from the lungs towards other organs has been undertaken by Oberdörster et al [ 19 ]. In inhalation experiments with rats, using 13C-labelled particles, they found that nano-sized particles (25 nm) were present in several organs 24 hours after exposure. The most extraordinary finding was the discovery of particles in the central nervous system (CNS). The authors examined this phenomenon further and found that particles, after being taken up by the nerve cells, can be transported via nerves (in this experiment via the olfactory nerves) at a speed of 2.5 mm per hour [ 56 ]. Passage of solid material from the pulmonary epithelium to the circulation seems to be restricted to nanoparticles. The issue of particle translocation still need to be clarified: both the trans-epithelial transport in the alveoli and the transport via nerve cells. Thus, the role of factors governing particle translocation such as the way of exposure, dose, size, surface chemistry and time course should be investigated. For instance, it would be also very important to know how and to what extent lung inflammation modulates the extra-pulmonary translocation of particles. 3.5 Fibre bio-persistence Long non-phagocytizable fibres (in humans longer than 20 micron) will not be effectively cleared from the respiratory tract. The main determinants of fibre bio-persistence are species specific physiological clearance and fibre specific bio-durability (physical-chemical processes). In the alveoli the rate at which fibres are physically cleared depends on the ability of alveolar macrophages to phagocytose them. Macrophages containing fibres longer than their own diameter may not be mobile and be unable to clear the fibres from the lung. The bio-durability of a fibre depends on dissolution and leaching as well as mechanical breaking and splitting. Long fibres in the lung can disintegrate, leading to shorter fibres that can be removed by the macrophages. Bio-persistent types of asbestos, where breakage occurs longitudinally, result in more fibres of the same length but smaller diameter. Amorphous fibres break perpendicular to their long axis [ 60 , 61 ], resulting in fibres that can be engulfed by the macrophages. It is self-evident that the slower the fibres are cleared (high bio-persistence), the higher is the tissue burden and the longer the fibres reside in a tissue the higher is the probability of an adverse response. A milestone was set by Stanton et al [ 62 , 63 ] who undertook a series of experiments with 17 samples of carefully sized fibrous glass. They found that for mesothelioma induction in rats, the peak activity was in the fibres greater than 8 micron in length and less than 1.3 micron in diameter. These findings are known as the "Stanton hypothesis". However these results do not strictly indicate that all fibres longer than the lower threshold are equally active or that shorter fibres are not, although fibres less than 5 micron in length did not appear to contribute to lung cancer risk in exposed rats [ 64 ]. Risk appears to increase with length, with fibres more than 40 micron in length imposing the highest risk. For the recent review see Schins [ 65 ]. The bio-durability of fibres with a diameter < 100 nm will probably not differ from larger inhalable fibres. Therefore, great caution must be taken in case of the contact with nano-fibres, Bio-durability tests must be performed before releasing any products containing them. Carbon nanotubes, which are of high technical interest, are one of the materials which need to be tested in depth concerning bio-persistence and cancer risk. The first toxicological studies indicated that carbon nanotubes can be a risk for human health [ 22 - 24 ], while exposure assessment did indicate that these materials are probably not inhaled [ 25 ]. 4. Intestinal tract Already in 1926 it was recognised by Kumagai [ 66 ] that particles could translocate from the lumen of the intestinal tract via aggregations of intestinal lymphatic tissue (Peyer's patches (PP)), containing M-cells (specialised phagocytic enterocytes). Particulate uptake happens not only via the M-cells in the PP and the isolated follicles of the gut-associated lymphoid tissue, but also via the normal intestinal enterocytes. There have been a number of excellent reviews on the subject of intestinal uptake of particles [ 51 , 66 ]. Uptake of inert particles has been shown to occur trans-cellularly through normal enterocytes and PP via M-cells, and to a lesser extent across para-cellular pathways [ 67 ]. Initially it was assumed that the PP did not discriminate strongly in the type and size of the absorb particles. Later it has been shown that modified characteristics, such as particle size [ 68 ] the surface charge of particles [ 69 ], attachment of ligands [ 70 , 71 ] or coating with surfactants [ 72 ], offers possibilities of site-specific targeting to different regions of the gastro intestine tract (GIT), including the PP [ 73 ]. The kinetics of particle translocation in the intestine depends on diffusion and accessibility through mucus, initial contact with enterocyte or M-cell, cellular trafficking, and post-translocation events. Charged particles, such as carboxylated polystyrene nanoparticles [ 69 ] or those composed of positively charged polymers exhibit poor oral bioavailability through electrostatic repulsion and mucus entrapment. Szentkuti [ 74 ] determined the rate of particle diffusion across the mucus layer to the enterocyte surface with respect to both size and surface charge of the particles. In brief, Szentkuti [ 74 ] observed that cationic nanometer-sized latex particles became entrapped in the negatively charged mucus, whereas repulsive carboxylated fluorescent latex nanoparticles were able to diffuse across this layer. The smaller the particle diameter the faster they could permutate the mucus to reach the colonic enterocytes; 14 nm diameter permeated within 2 min, 415 nm particles took 30 min, while 1000-nm particles were unable to translocate this barrier. Within, the time of the experiment (30 min) none of the particles was endocytosed by the enterocytes despite the fact that the latex nanoparticles preferentially bound the cell surface more strongly than the mucus. After a longer time window (oral gavage for several days) a sparse accumulation of charged particulates in the lamina propria (connective tissue under the epithelia) was found compared to uncharged latex nanoparticles in the same size range [ 69 ]. Particulates, once in the sub-mucosal tissue, are able to enter both lymphatic and capillaries. Particles entering the lymphatic are probably important in the induction of secretory immune responses while those which enter the capillaries become systemic and can reach different organs. In one study [ 75 ], the body distribution after translocation of polystyrene particles was examined in some detail. Polystyrene spheres (ranging from 50 nm to 3 micron) were fed by gavage to female Sprague Dawley rats daily for 10 days at a dose of 1.25 mg/kg. As much as 34 % and 26% of the 50 and 100 nm particles were absorbed respectively. Those larger than 300 nm were absent from blood. No particles were detected in heart or lung tissue. 4.1 Intestinal Translocation and Disease Crohn's disease is characterised by transmural inflammation of the gastrointestinal tract. It is of unknown aetiology, but it is suggested that a combination of genetic predisposition and environmental factors play a role. Particles (0.1–1.0 micron) are associated with the disease and indicated as potent adjuvants in model antigen-mediated immune responses. In a double-blind randomised study, it has been shown that a particle low diet (low in calcium and exogenous microparticles) alleviates the symptoms of Crohn's disease [ 76 ]. Although there is a clear association between particle exposure and uptake and Crohn's disease, little is known of the exact role of the phagocytosing cells in the intestinal epithelium. It has been suggested that the disruption of the epithelial barrier function by apoptosis of enterocytes is a possible trigger mechanism for mucosal inflammation. The patho-physiological role of M cells is unclear; e.g., it has been found that in Crohn's disease M cells are lost from the epithelium. Other studies found that material uptake (endocytose) capacity of M cells is induced under various immunological conditions, e.g. a greater uptake of particles (0.1 micron, 1 micron and 10 micron diameter) has been demonstrated in the inflamed colonic mucosa of rats compared to non-ulcerated tissue [ 77 , 78 ] and inflamed oesophagus [ 79 ]. Diseases other than of gut origin also have marked effects on the ability of GIT to translocate particles. The absorption of 2-micron polystyrene particles from the PP of rats with experimentally induced diabetes is increased up to 100-fold (10% of the administered dose) compared to normal rats [ 80 ]. However, the diabetic rat displayed a 30% decrease in the systemic distribution of the particles. One possible explanation for this discrepancy is the increased density of the basal lamina underlying the GI mucosa of diabetic rats that may impede particle translocation into deeper villous regions. This uncoupling between enhanced intestinal absorption and reduced systemic dissemination has also been observed in dexamethasone treated rats [ 81 ]. From the literature cited above it is clear that engineered nanoparticles can be taken up via the intestinal tract. In general the intestinal uptake of particles is better understood and studied in more detail than pulmonary and skin uptake. Because of this advantage it is maybe possible to predict the behaviour of some particles in the intestines but precaution should be taken. For those nanoparticles designed to stabilise food or to deliver drug via intestinal uptake other, more demanding, rules exist and should be followed before marketing these compounds. 5. Skin Skin is an important barrier, protecting against insult from the environment. The skin is structured in three layers: the epidermis, the dermis and the subcutaneous layer. The outer layer of the epidermis, the stratum corneum (SC), covers the entire outside of the body and only contains dead cells, which are strongly keratinized. For most chemicals the SC is the rate-limiting barrier to percutaneous absorption (penetration). The skin of most mammalian species is, on most parts of the body, covered with hair. At the sites, where hair follicles grow, the barrier capacity of the skin differs slightly from the "normal" stratified squamous epidermis. Most studies concerning penetration of materials into the skin have focussed on whether or not drugs penetrate through the skin using different formulations containing chemicals and/or particulate materials as a vehicle. The main types of particulate materials commonly used are: liposomes; solid poorly soluble materials such as TiO2 and polymer particulates and submicron emulsion particles such as solid lipid nanoparticles. The penetration of these particulate carriers has not been studied in detail. TiO2 particles are often used in sunscreens to absorb UV light and therefore to protect skin against sunburn or genetic damage. It has been reported by Lademann et al in [ 82 ] that micrometer-sized particles of TiO2 get through the human stratum corneum and even into some hair follicles – including their deeper parts. However, the authors did not interpret this observation as penetration into living layers of the skin, since this part of the follicular channel (the acroinfundibulum) is covered with a horny layer barrier too [ 82 ]. A different interpretation has been suggested in a recent review by Kreilgaard [ 83 ], who argued that "very small titanium dioxide particles (e. g. 5–20 nm) penetrate into the skin and can interact with the immune system". Tinkle et al [ 84 ] demonstrated that 0.5- and 1.0 micron particles, in conjunction with motion, penetrate the stratum corneum of human skin and reach the epidermis and, occasionally, the dermis. The authors hypothesised that the lipid layers within the cells of the stratum corneum form a pathway by which the particles can move [ 85 ] into the skin and be phagocytized by the Langerhans cells. In this study the penetration of particles is limited to particle diameter of 1 micron or less. Nevertheless, other studies reported penetration through the skin using particles with diameters of 3–8 micron [ 86 , 87 , 82 ] but only limited penetration was found often clustered at the hair follicle (see above). Penetration of non-metallic solid materials such as biodegradable poly(D,L-lactic-co-glycolic acid (PLGA) microparticles, 1 to 10 micron with a mean diameter of 4.61 ± 0.8 micron was studied after application on to porcine skin. The number of microparticles in the skin decreased with the depth (measured from the airside towards the subcutaneous layer). At 120 micron depth (where viable dermis present) a relatively high number of particles was found, at 400 micron (dermis) some micro-particles were still seen. At a depth of 500 micron no microparticles were found [ 88 ]. In the skin of individuals, who had an impaired lymphatic drainage of the lower legs, soil microparticles, frequently 0.4–0.5 micron but as larger particles of 25 micron diameter, were found in the in the dermis of the foot in a patient with endemic elephantiasis. The particles are seen to be in the phagosomes of macrophages or in the cytoplasm of other cells. The failure to conduct lymph to the node produces a permanent deposit of silica in the dermal tissues (a parallel is drawn with similar deposits in the lung in pneumoconiosis). This indicates that soil particles penetrate through (damaged) skin, most probably in every individual, and normally are removed via the lymphatic system [ 89 , 90 ]. Liposomes penetrate the skin in a size dependent manner. Micro-sized, and even submicron sized, liposomes do not easily penetrate into the viable epidermis, while liposomes with an average diameter of 272 nm can reach into the viable epidermis and some are found in the dermis. Smaller sized liposomes of 116 and 71 nm were found in higher concentration in the dermis. Emzaloid™ particles, a type of submicron emulsion particle such as liposomes and nonionic surfactant vesicles (niosomes), with a diameter of 50 nm to 1 micron, were detected in the epidermis in association with the cell membranes after application to human skin [ 91 ]. The authors suggested that single molecules, which make up the particles, may penetrate the intercellular spaces and, at certain regions in the stratum corneum, are able to accumulate and reform into micro spheres. In a subsequent experiment, it was shown that the used formulation allowed penetration of the spheres into melanoma cells, even to the nucleus [ 92 ]. A recent review by Hostynek [ 93 ] stated that the uptake of metals through the skin is complex, because of both exogenous factors (e.g. dose, vehicle, protein reactivity, valence) and endogenous factors (e.g. age of skin, anatomical site, homeostatic control). Attempts to define rules governing skin penetration to give predictive quantitative structure-diffusion relationships for metallic elements for risk assessment purposes have been unsuccessful, and penetration of the skin still needs to be determined separately for each metal species, either by in vitro or in vivo assays. Only limited literature on nanoparticles penetrating the skin is available, but some conclusions can already be drawn. Firstly, penetration of the skin barrier is size dependent, nano-sized particles are more likely to enter more deeply into the skin than larger ones. Secondly, different types of particles are found in the deeper layers of the skin and at present it is impossible to predict the behaviour of a particle in the skin. And finally, materials, which can dissolve or leach from a particle (e.g. metals), or break into smaller parts (e.g. Emzaloid™ particles), can possibly penetrate the skin. We did not find any direct indication that particles, that had penetrated the skin, also entered the systemic circulation. The observation that particles in the skin can be phagositized by macrophages, Langerhans cells or other cells is a possible road towards skin sensitisation. Tinkle et al [ 84 ] have shown that topical application of beryllium, to C3H mice, generated beryllium-specific sensitisation. These data are consistent with the development of a hapten-specific, cell-mediated immune response. 5.1 Mechanical irritation of skin Glass fibres and Rockwool fibres are widely distributed man-made mineral fibres because of their multiple applications, mainly as insulation materials, which have become important for replacing asbestos fibres. In contact with the skin, these fibres can induce dermatitis through the mechanical irritation. Why these fibres are such strong irritant has not been examined in detail. In occlusion irritant patch tests in humans it was found that Rockwool fibres with a diameter of 4.20 ± 1.96 micron were more irritating than those with a mean diameter of 3.20 ± 1.50 micron. The fact that "small" fibres can cause strong skin irritation has been known for a long time, e.g. itching powder. It is also commonly accepted that some types of man made fibres can easily induce non-allergic dermatitis. Although this is common knowledge, it is not clear what makes these fibres irritants. In search for reports on skin irritation caused by fibres with a diameter of < 100 nm no information could be found, indicating that more research is needed. 6. Body distribution and systemic effects of particulates The body distribution of particles is strongly dependent on their surface characteristics. For example, coating poly(methyl methacrylate) nanoparticles with different types and concentrations of surfactants significantly changes their body distribution [ 116 ]. Coating these nanoparticles with ≥ 0.1 % poloxamine 908 reduces their liver concentration significantly (from 75 to 13 % of total amount of particles administrated) 30 min after intravenous injection. Another surfactant, polysorbate 80, was effective above 0.5%. A different report [ 94 ] shows that modification of the nanoparticle surface with a cationic compound, didodecyldimethylammonium bromide (DMAB), facilitates the arterial uptake 7–10-fold. The authors noted that the DMAB surface modified nanoparticles had a zeta potential of +22.1 +/- 3.2 mV (mean +/- sem, n = 5) which is significant different from the original nanoparticles which had a zeta potential of -27.8 +/- 0.5 mV (mean +/- sem, n = 5). The mechanism for the altered biological behaviour is rather unclear, but surface modifications have potential applications for intra-arterial drug delivery. Oral uptake (gavage) of polystyrene spheres of different sizes (50 nm to 3 micron) in female Sprague Dawley rats (for 10 days at a dose of 1.25 mg/kg/day) resulted in systemic distribution of the nanoparticles. About 7% (50 nm) and 4% (100 nm), was found in the liver, spleen, blood and bone marrow. Particles larger than 100 nm did not reach the bone marrow and those larger than 300 nm were absent from blood. No particles were detected in heart or lung tissue [ 75 ]. Irrespective of the uptake route, the body distribution of particles, is most dependent on the surface characteristics and the size of the particles. It is an important issue in drug-design in order to help to deliver medication to the right target. In unintentional uptake of nanoparticles these characteristics can strongly influence the accumulation of a specific type of particle in the particular body site. 6.1 Nanoparticles, thrombosis and lung inflammation Epidemiological studies have reported a close association between particulate air pollution and cardiovascular adverse effects such as myocardial infarction [ 95 ]. The latter results from rupture of an atherosclerotic plaque in the coronary artery, followed by rapid thrombus growth caused by exposure of highly reactive subendothelial structures to circulating blood, thus leading to additional or complete obstruction of the blood vessel. Nemmar et al [ 96 ] studied the possible effects of particles on haemostasis, focusing on thrombus formation as a relevant endpoint. Polystyrene particles of 60 nm diameter (surface modifications: neutral, negative or positive charged) have a direct effect on haemostasis by the intravenous injection. Positively charged amine-particles led to a marked increase in prothrombotic tendency, resulting from platelet activation. A similar effect could be obtained after the intratracheal administration of these positively charged polystyrene particles, which also caused lung inflammation [ 97 ]. It is important to indicate that the pulmonary instillation of larger (400 nm) positive particles caused a definite pulmonary inflammation (of similar intensity to 60 nm particles), but they did not lead to a peripheral thrombosis within the first hour of exposure. This lack of effect of the larger particles on thrombosis, despite their marked effect on pulmonary inflammation, suggests that pulmonary inflammation by itself was insufficient to influence peripheral thrombosis. Consequently, the effect found with the smaller, ultrafine particles is most probably due, at least in part, to their systemic translocation from the lung into the blood. Pollutant particles such as diesel exhaust particles (DEP), may cause a marked pulmonary inflammation within an hour after their deposition in the lungs. Moreover, intratracheal instillation of DEP promotes femoral venous and arterial thrombosis in a dose-dependent manner, already starting at a dose of 5 μg per hamster (appr. 50 μg/kg). Subsequent experiments showed that prothrombotic effects persisted at 6 h and 24 h after instillation (50 μg/animal) and confirmed that peripheral thrombosis and pulmonary inflammation are not always associated [ 97 ]. Solid inhaled particles are a risk for those who suffer from cardiovascular disease. Experimental data indicate that many inhaled particles can affect cardiovascular parameters, via pulmonary inflammation. Nano-sized particles, after passage in the circulation, can also play a direct role in e.g. thrombogenisis. Epidemiologic studies have provided valuable information on the adverse health effects of particulate air pollution in the community, indicating that nanoparticles act as an important environmental risk factor for cardiopulmonary mortality. Particle-induced pulmonary and systemic inflammation, accelerated atherosclerosis, and altered cardiac autonomic function may be part of the patho-physiological pathways, linking particulate air pollution with cardiovascular mortality. Also, it has been shown that particles deposited in the alveoli lead to activation of cytokine production by alveolar macrophages and epithelial cells and to recruitment of inflammatory cells. An increase in plasma viscosity, fibrinogen and C-reactive protein has been observed in samples of randomly selected healthy adults in association with particulate air pollution [ 95 , 98 , 99 ]. 6.2 Nanoparticles and cellular uptake A number of reports on cellular uptake of micro- and nano- sized particles has been published. Reports on particle uptake by endothelial cells [ 100 , 101 ], pulmonary epithelium [ 102 , 79 , 103 , 59 ], intestinal epithelium [ 51 , 79 ] alveolar macrophages [ 104 - 107 , 57 ], other macrophages [ 89 , 108 , 76 , 109 ], nerve cells [ 110 ] and other cells[ 111 ] are available. This is an expected phenomenon for phagocytic cells (macrophages) and cells that function as a barrier and/or transport for (large) compounds. Except for macrophages, the health effects of cellular uptake of nanoparticles have not been studied in depth. 6.3 Nanoparticles and the blood-brain barrier One of the promising alleys of nanotechnology is organ- or cell- specific drug delivery mediated by nanoparticles [ 112 - 114 ]. It is expected that transport of nanoparticles across the blood-brain barrier (BBB) is possible by either passive diffusion or by carrier-mediated endocytosis. Coating of particles with polysorbates (e.g. polysorbate-80) results in anchoring of apolipoprotein E (apo E) or other blood components. Surface modified particles seem to mimic LDL particles and can interact with the LDL receptor leading to uptake by endothelial cells. Hereafter, the drug (which was loaded in the particle) may be released in these cells and diffuse into the brain interior or the particles may be trans-cytosed. Also, other processes such as tight junction modulation or P-glycoprotein (Pgp) inhibition also may occur [ 115 ]. Oberdörster et al 2002 reported the translocation of inhaled nanoparticles via the olfactory nerves [ 56 ]. Drug delivery systems crossing the BBB are certainly welcome, but this also implicates that unintended passage through the BBB is possible; therefore good safety evaluations are needed. 6.4. Nanoparticles and oxidative stress It has been shown that nanoparticles, that enter the liver, can induce oxidative stress locally. A single (one day; 20 and 100 mg/kg) and repeated (14 days) intravenous administration of poly-isobutyl cyanoacrylate (PIBCA, a biodegradable particle) or polystyrene (PS, not biodegradable) nanoparticles induced a depletion of reduced glutathione (GSH) and oxidised glutathione (GSSG) levels in the liver, as well as inhibition of superoxide dismutase (SOD) activity and a slight increase in catalase activity. The nanoparticles did not distribute in the hepatocytes, implicating that the oxidative species most probably were produced by activated hepatic macrophages, after nanoparticle phagocytosis. Uptake of polymeric nanoparticles by Kupffer cells in the liver induces modifications in hepatocyte antioxidant systems, probably due to the production of radical oxygen species [ 108 ]. We have discussed above that nano-sized particles in the lung can induce, via the pulmonary inflammatory response as well as via spontaneously surface related reactions, oxidative stress. Besides pulmonary studies, not many have studied particle-induced oxidative stress in tissues. However, the authors [ 108 ] reported that the depletion in glutathione was not sufficient enough to initiate significant hepatocytic damage (no lipid peroxidation). It needs to be stressed that long-term studies are needed to prove the safe use of these nanoparticles because chronic depletion of the anti-oxidant defence can lead to severe health problems. 7. Differences in conditions between the lung and intestinal tract Although the contact with nanomaterials in the lungs and intestinal tract shows many similarities important differences between inhalation and ingestion of nanomaterials exist from the toxicological point of view. In the intestinal tract a complex mix of compounds – such as secreted enzymes, ingested food, bacteria of the gut flora, etc – is present, which can interact with the ingested nanomaterial. Non-specific interaction often reduces the toxicity of the ingested material. It has been described that in vitro particles are less cytotoxic when dosed in a medium with high protein content. In the lungs, mucus or surfactant is present, in which antioxidants are present, but these can be easily neutralised when a high number of oxidative compounds is inhaled. The transit through the intestinal tract is a relatively fast process, the continuous decay and renewal of the epithelium makes sure that nanomaterials will not remain long in the intestinal tract. The presence of solid material in the lumen of the intestines will not automatically induce an inflammatory response. Inhaled materials < 10 micron and > 5 micron will not enter the alveolar spaces of the lungs, and therefore these will be cleared easily in healthy persons via the muco-ciliary escalator. Particles that are smaller than 5 micron will deposit in the alveolar space via Brownian movement. In the alveoli, water insoluble materials can only be removed via phagocytosis by macrophages or other cells, or via transportation through the epithelium to the interstitium or systemic circulation. These processes are often accompanied by the onset of (persistent) inflammation. The particles themselves can – depending on the physical-chemical characteristics of the material – remain for a long period in the alveoli. In the intestinal tract, the ingested materials are stressed from acidic (stomach) to basic conditions. The shift in pH markedly changes the solubility and the ionic state of the material via changing the surface characteristics. In the lungs, the milieu of the lumen is more constant. 8. Conclusions Particles in the nano-size range can certainly enter the human body via the lungs and the intestines; penetration via the skin is less evident. It is possible that some particles can penetrate deep into the dermis. The chances of penetration depend on the size and surface properties of the particles and also on the point of contact in the lung, intestines or skin. After the penetration, the distribution of the particles in the body is a strong function of the surface characteristics of the particles. A critical size might exist beyond which the movement of the nanoparticles in parts of the body is restricted. The pharmaco-kinetic behaviour of different types of nanoparticles requires detailed investigation and a database of health risks associated with different nanoparticles (e.g. target organs, tissue or cells) should be created. The presence of the contaminates, such as metal catalysts present in nanotubes, and their role in the observed health effects should be considered along with the health effect of the nanomaterials. The increased risk of cardiopulmonary diseases requires specific measures to be taken for every newly produced nanoparticle. There is no universal "nanoparticle" to fit all the cases, each nanomaterial should be treated individually when health risks are expected. The tests currently used to test the safety of materials should be applicable to identify hazardous nanoparticles. Proven otherwise, it would be a challenge for industry, legislators and risk assessors to construct a set of high throughput and low cost tests for nanoparticles without reducing the efficiency and reliability of the risk assessment. Nanoparticles designed for drug delivery or as food components need special attention. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544578.xml |
521493 | Lycopene from two food sources does not affect antioxidant or cholesterol status of middle-aged adults | Background Epidemiological studies have reported associations between reduced cardiovascular disease and diets rich in tomato and/or lycopene. Intervention studies have shown that lycopene-containing foods may reduce cholesterol levels and lipid peroxidation, factors implicated in the initiation of cardiovascular disease. The objective of this study was to determine whether consumption of lycopene rich foods conferred cardiovascular protection to middle-aged adults as indicated by plasma lipid concentrations and measures of ex vivo antioxidants. Methods Ten healthy men and women consumed a low lycopene diet with no added lycopene (control treatment) or supplemented with watermelon or tomato juice each containing 20 mg lycopene. Subjects consumed each treatment for three weeks in a crossover design. Plasma, collected weekly was analyzed for total cholesterol, high density lipoprotein cholesterol (HDL-C) and triglyceride concentrations and for the antioxidant biomarkers of malondialdehyde formation products (MDA), plasma glutathione peroxidase (GPX) and ferric reducing ability of plasma (FRAP). Data were analyzed using Proc Mixed Procedure and associations between antioxidant and lipid measures were identified by Pearson's product moment correlation analysis. Results Compared to the control diet, the lycopene-containing foods did not affect plasma lipid concentrations or antioxidant biomarkers. Women had higher total cholesterol, HDL-C and triglyceride concentrations than did the men. Total cholesterol was positively correlated to MDA and FRAP while HDL-C was positively correlated to MDA and GPX. GPX was negatively correlated to triglyceride concentration. Conclusions The inclusion of watermelon or tomato juice containing 20 mg lycopene did not affect plasma lipid concentrations or antioxidant status of healthy subjects. However, plasma cholesterol levels impacted the results of MDA and FRAP antioxidant tests. | Background Watermelons and tomatoes are good sources of the carotenoid lycopene [ 1 , 2 ]. However, bioavailability of lycopene is not directly related to plant content, and depends in a large part upon plant matrix effects. In tomatoes, heat processing and homogenization breaks protein-carotenoid complexes, releases lycopene from cell wall linkages and improves human uptake of this compound [ 3 - 6 ], while heat processing is not necessary for adequate uptake of lycopene from watermelon juice [ 7 ]. Extracts of both foods exhibit antioxidant activity in vitro and function is attributed to lycopene since isolated lycopene demonstrates strong oxygen and peroxy radical scavenging properties [ 8 - 10 ]. Recent epidemiological studies have linked reductions in risks of cardiovascular disease with diets rich in lycopene containing foods. These reductions in risk have been primarily attributed to the antioxidant properties of lycopene [ 11 , 12 ]. Improved antioxidant parameters of lymphocytes have been reported in clinical trials that supplemented diets with 16.5 mg and 40 mg /day of lycopene from tomato puree and tomato juice, respectively [ 13 , 14 ]. Other clinical trials have shown reductions in low-density lipoprotein (LDL) oxidation resulting from lycopene supplementation [ 15 - 17 ]. LDL contains unsaturated fatty acids and can be oxidized by free radicals and peroxidizing agents. Since lycopene is primarily attached to LDL in plasma, it may protect against atherosclerosis through inhibition of lipid peroxidation and foam cell production [ 12 , 18 ]. Other studies have assessed response of plasma lipids to lycopene-rich diets. In one study, six healthy men were supplemented with 60 mg/day for three months with tomato lycopene (LycoRed) with a 14% reduction in LDL-C and no change in HDL-C [ 19 ]. Researchers concluded that lycopene was involved in controlling cholesterol synthesis and found the same results in a macrophage cell study [ 19 ]. It is not known if other lycopene containing foods can act ex vivo as antioxidants or alter cholesterol levels. The objectives of this study were to compare the ability of two lycopene containing foods, tomato and watermelon to provide cardiovascular protection to middle-aged adults by measuring changes in cholesterol levels and antioxidant ex vivo biomarkers. Methods Experimental Design Samples for this study came from a larger study, which has been reported in detail [ 7 ]. This study was a diet-controlled, repeated measures crossover design with ten healthy non-smoking subjects, five men (average age 49 years) and five women (average age 51 years) recruited from the Beltsville, MD area (Table 1 ). In addition to a base diet, which provided 34% of energy from fat and minimal amounts of lycopene, subjects were randomly assigned to receive three dietary treatments for 3 weeks each: 1) control (no added lycopene); 2) 20.1 mg lycopene per day from watermelon juice; and 3) 18.4 mg lycopene per day from tomato juice. All subjects followed a low-lycopene diet for two weeks before the first treatment and during the four-week washout periods between treatments. Total study duration was 19 weeks. During treatment periods, all meals were prepared and consumed Monday through Friday at the Beltsville Human Nutrition Research Center's Human Studies Facility, and weekend meals were packed for off-site consumption. Blood was drawn from fasted subjects before treatment (the day before the start of study and on the first day of the study), prior to treatment and weekly during treatment. Plasma was separated from whole blood by centrifugation and stored at -80°C until analyzed for cholesterol and antioxidant activity. Table 1 Description of human clinical study participants. Gender N Age (Range) yr BMI (Range) Kg/m 2 Men 5 49 (43–68) 26.3 (23.0–29.5) Women 5 51 (35–63) 29.1 (23.5–34.5) Juice Treatments Watermelon juice for the study was prepared at a pilot plant at the USDA Citrus and Subtropical Products Laboratory, Winter Haven, FL without heat treatment as previously described [ 7 ]. Canned commercial tomato juice (Campbell's, Camden, NJ) was used for the tomato intervention. Juices were analyzed for carotenoid content using established extraction procedures with reversed phase HPLC with photo diode array detector (Waters Corp, Franklin, MA) [ 7 ]. For watermelon treatment, subjects were given one bottle of juice (260 g each) at breakfast, lunch and dinner, which provided daily totals of 20.1 mg lycopene, 0.90 mg phytoene, 0.45 mg phytofluene and 2.5 mg beta carotene. The juice contained 94% trans lycopene and 6% cis isomers, primarily 5- cis and 13- cis with minimal amounts of other cis isomers [ 7 ]. For tomato juice treatment, subjects were given one serving (122 g each) at breakfast and dinner, which provided daily totals of 18.4 mg lycopene, 2.1 mg phytoene, 1.1 mg phytofluene and 0.6 mg beta carotene with 89% of the lycopene as trans lycopene and 10.8% cis isomers, primarily identified as 5- cis , 9- cis , 13- cis , and 15- cis , and minimal amounts of other cis isomers [ 7 ]. Cholesterol Analysis Plasma samples were thawed on ice for four hours then mixed by vortexing, prior to preparing for assays. Serum total cholesterol and triglyceride concentrations were determined enzymatically using kits from Roche Diagnostics (Sommerville, NJ). Serum HDL-cholesterol was determined by a direct method (Unimate HDL Direct ; Roche Diagnostics, Indianapolis, IN) that utilizes the combined action of polymers, polyanions, and detergent to solubilize cholesterol from HDL but not from VLDL, LDL, and chylomicrons as previously described [ 20 ]. Analysis was performed on a Cobas-Fara II Clinical Analyzer (Montclair, NJ) using commercially available calibrators and quality control standards (Roche Diagnostics, Indianapolis, IN). Plasma Glutathione Peroxidase Assay Plasma from subjects was analyzed for plasma glutathione peroxidase using an ELISA kit (OXIS Internatl., Portland, OR). Two replicates per sample of 20 μl of plasma were diluted 1:25 with TRIS-HCl buffer then pippetted into pre-coated polyclonal antibodies microplate wells specific for human plasma glutathione peroxidase (GPX). The amount of enzyme present was determined by reaction with para-nitrophenyl-phosphate and was read using a microplate reader at 405 nm (Elx 808 Ultra Microplate Reader, Bio-Tek Instruments Inc., Winooski, VT). The concentration of plasma GPX was determined from a standard curve for each plate using five dilutions of GPX standard. Plasma lipid peroxidation Malondialdehyde compounds were determined colorimetrically using a commercial kit specific for measuring free and total malondialdehyde compounds (OXIS Internatl., Portland, OR). Two replicates per sample of 210 μl of plasma were added to each test tube with 11 μl of 500 mM butylated hydroxytoluene and 5.3 μl of concentrated hydrochloric acid. Tubes were capped, mixed then incubated at 60°C for 80 minutes, cooled to room temperature and 680 μl of N-methyl-2-phenylindole in acetonitrile was added. Then tubes were mixed, and centrifuged at 13,000 g for 5 minutes. New tubes were prepared and 660 μl of clear supernatant was added with 115 μl of concentrated HCl. Tubes were capped, mixed and incubated at 45°C for 60 minutes. Samples were centrifuged at 13,000 g for 5 min and the supernatant was read on a spectrophometer at 575 nm. Concentration of samples was determined using a five point standard curve. Ferric reducing ability of plasma assay This assay was conducted according to previously published methods [ 1 ]. In brief, three reagents were used: 1) sodium acetate, acetic acid buffer (pH 3.6); 2)10 mmol/L solution of 2, 4,6-tripyridyl-s-triazine in a 40 mmol/L solution of hydrochloric acid (Sigma, St. Louis, MO); and 3) 20 mmol/L solution of ferric chloride hexahydrate prepared in double deionized water. The FRAP reagent was prepared daily with 25 ml of reagent one, 2.5 ml reagent two and three that were heated to 37°C before using [ 21 ]. The assay was conducted with 10 uL of plasma that was diluted with 30 μl of ddi water. Sample was added to reagent in cuvettes with an autosampler and then read on a COBAS FARA II spectrofluorometric centrifugal analyzer (Roche, Montclair, NJ) at 593 nm at four minutes. FRAP values were determined from a five point curve using a trolox (vitamin E analog) standard. Standard curves were run after every 90 samples. Experimental procedures for the clinical trial were approved by the Institutional Review Board at the Johns Hopkins University Bloomberg School of Hygiene and Public Health; subjects gave their written informed consent to participate. The plasma cholesterol and antioxidant studies were approved by the Institutional Review Board at Oklahoma State University, Stillwater, OK. Data were analyzed using Proc Mixed Procedure and mean separation was performed using LSMEANS, correlation analysis was performed using Spearman's Correlation Coefficient Analysis (SAS Statistical Analysis Software, version 8.2, SAS Institute, Cary, NC). Results Because there were significant four way interactions with gender × intervention period × treatment × weeks with MDA, FRAP, GPX and cholesterol analysis, trends by treatment, intervention period or week of treatment were not seen. Supplementing the diet with 20 mg/day of lycopene of either food did not change the plasma antioxidant status of the subjects and values ranged from 0.66–2.20, 540–1094, and 1296–2596 :mol/L for MDA, FRAP and GPX respectively. These levels are similar to levels reported for healthy subjects in other studie [ 22 , 23 ]. Intervention with 20 mg of lycopene to the diet of subjects did not alter their total cholesterol, HDL-C or triglyceride status. However, there were gender differences and the women had higher average levels of plasma triglycerides, total cholesterol and HDL-C than men (Figure 1 ). The higher cholesterol levels for women compared to men in this study were not unusual since women in this age range often have higher cholesterol levels than men, a phenomenon related to decreased estrogen production [ 24 , 25 ]. In this study, menopausal information was not recorded. Figure 1 Mean plasma total cholesterol, triglycerides, and high density lipoprotein (HDL) cholesterol (mg/dl) of 5 men and 5 women supplemented for 3 week with no lycopene (control), 20 mg lycopene from watermelon juice and 20 mg lycopene from tomato juice. *Represents significance p < 0.05. There was a significant positive correlation between each pair of total cholesterol and MDA and MDA and FRAP and between HDL-C and MDA and HDL-C and GPX. A significant negative correlation was found between triglycerides and GPX (Table 2 ). Table 2 Spearman's Correlation Coefficients between antioxidant tests of malondialdehyde (MDA) ferric reducing ability of plasma (FRAP), and plasma glutathione peroxidase (GPX) and cholesterol measurements. Variable MDA FRAP GPX Total cholesterol 0.547** 0.325** 0.003 HDL-C 0.563** 0.059 0.294** Triglycerides 0.219 0.037 -0.229* MDA . 0.474** 0.180 FRAP 0.474** . 0.077 Correlations significant at the 0.05* and 0.01** level. Because of the correlation between cholesterol concentrations and antioxidant analysis, a preliminary analysis of data was conducted to determine if cholesterol levels impacted antioxidant results. Subjects were separated into two groups based upon baseline concentrations of plasma triglycerides, total cholesterol and LDL-C above or below 200, 180 and 160, respectively. Five subjects, two men and three women, fit the criteria of moderately hypercholesterolemic (Table 3 ). Table 3 Separation of subjects by baseline cholesterol levels from a watermelon and tomato juice lycopene intervention study, n = 5 for each group. Cholesterol Group Total Cholesterol mg/dl Triglycerides mg/dl HDL-Cmg/dl 1 229.3 ± 4.9 190.9 ± 12.8 59.1 ± 2.9 2 176.6 ± 2.5 129.7 ± 5.2 46.9 ± 2.1 Data represents mean ± SE Analyses showed an interaction of cholesterol level × treatment period × treatment factor for MDA and FRAP analysis. Higher MDA and FRAP levels were found in the group having higher cholesterol levels compared to the other group (Table 4 ). No trend with cholesterol level and glutathione peroxidase was found. Table 4 Total, triglyceride, high density lipoprotein (HDL-C cholesterol and antioxidant analysis of malondialdehyde (MDA), glutathione peroxidase (GPX) and ferric reducing ability of of plasma (FRAP) after 4 week lycopene depletion and three weeks of intervention with watermelon and tomato juice (20 mg lycopene/day). Subjects were separated into 2 groups based upon cholesterol levels (see Table 3). Cholesterol Group Analysis Depletion SE Control SE Watermelon SE Tomato SE Total Cholesterol (mg/dl) 220.9 ± 9.5 223.4 ± 7.9 224.6 ± 8.2 233.6 ± 6.2 Triglycerides (mg/dl) 185.9 ± 16.9 181.7 ± 16.9 198.9 ± 18.3 174.7 ± 15.6 1 HDL-C (mg/dl) 56.9 ± 5.15 58.65 ± 4.31 58.00 ± 5.96 59.38 ± 4.55 MDA (umol/L) 1.21 ± 0.11 1.12 ± 0.11 1.15 ± 0.12 1.37 ± 0.11 GPX (umol/L) 2728 ± 219 2728 ± 222 2263 ± 169 2574 ± 187 FRAP (umol/L) 831.6 ± 24.9 871.7 ± 26.7 900.9 ± 25.2 861.6 ± 23.4 Total Cholesterol (mg/dl) 182.6 ± 4.2 173.3 ± 3.3 186.1 ± 6.4 173.1 ± 2.8 Triglycerides (mg/dl) 129.4 ± 8.3 128.7 ± 7.6 189.4 ± 4.9 135.4 ± 10.7 2 HDL-C (mg/dl) 42.5 ± 3.3 43.6 ± 2.8 44.7 ± 4.0 38.3 ± 2.8 MDA (umol/L) 0.53 ± 0.03 0.56 ± 0.04 0.48 ± 0.04 0.54 ± 0.03 GPX (umol/L) 2129 ± 151 2111 ± 154 2292 ± 168 2229 ± 160 FRAP (umol/L) 743.9 ± 33.6 762.9 ± 30.7 780.9 ± 32.4 756.7 ± 36.0 Discussion We found no improvement in the antioxidant status of healthy middle-aged adults supplemented with two lycopene-containing foods. In previous antioxidant studies, reduced lipid peroxidation was reported in subjects supplemented from one to four weeks with 5 to 45 mg lycopene containing tomato products [ 15 , 16 , 23 , 26 ]. However, in each of these studies, the diet was not controlled. When healthy elderly subjects in a diet controlled study were supplemented with 13.3 mg of tomato lycopene (LycoRed) for 12 weeks, lycopene intervention did not significantly change LDL oxidation, as measured by the rate of conjugated diene production [ 27 ]. The reports from lycopene intervention studies that measured FRAP activity are not in agreement. One study reported improvement in FRAP levels of plasma in subjects supplemented with tomato juice and olive oil [ 28 ], while two other tomato juice intervention studies reported no improvement in plasma antioxidant levels after lycopene supplementation as measured by Trolox equivalent antioxidant capacity (TEAC), radical trapping antioxidant parameter assay (TRAP), and FRAP [ 3 , 23 ]. Researchers in one study found that the FRAP assay was more accurate when measuring the antioxidant activity of water-soluble antioxidants [ 23 ]. They thought full expression of the antioxidant activity was not identified from lycopene in this assay since it is a lipophyllic compound. Curiously, both watermelon and tomato contain other water-soluble compounds that are reported to have antioxidant activity that reacts in vitro in the FRAP assay [ 9 , 29 ]. In this study, contribution of these water-soluble compounds in changes in plasma FRAP activity with either food intervention compared to the control was not found. Unlike a previous report by Fuhrman et al., neither lycopene intervention with watermelon nor tomato affected cholesterol levels [ 19 ]. Differences in results may have been due to lycopene dosage level. In that study [ 19 ] the subjects were supplemented with 60 mg/day for three months, however diet was not controlled. Fruits and vegetables are excellent sources of antioxidant compounds and the average American consumes only 1.5 and 3.1 servings per day [ 45 ]. In many of the studies where antioxidant protection with lycopene containing foods was reported, subjects consumed their normal diet that may or may not have met the recommended servings of fruits and vegetables [ 13 , 23 , 26 , 31 , 32 ]. Increasing fruit and vegetable consumption to 12 servings per day compared to 5.8 servings, without the addition of other diet interventions, reduced a biomarker of DNA oxidative damage (8-hydroxydeoxyguanosine) by 32% [ 33 ]. In a controlled trial where subjects were supplemented with tomato juice but restricted in total fruit and vegetable consumption and exposed to low levels of ozone, researchers found reduced DNA strand breaks compared to placebo controls [ 34 ]. Because this study controlled for other phytochemical containing fruits and vegetables, the DNA protection was attributed to tomato juice phytochemicals [ 34 ]. The positive correlation between total cholesterol and MDA antioxidant analysis has been reported in studies with hypercholesterolemic subjects compared to normocholesterolemic subjects [ 35 , 36 ]. The MDA assay measures lipid peroxidation products, and a higher level of lipids available to react with peroxidizing agents results in higher MDA values [ 36 , 37 ]. The trend correlating higher FRAP with higher cholesterol levels has not been previously reported. The significance of this trend is speculative, since the FRAP assay measures the oxidation and reduction potential of compounds based on the reduction of the ferric to ferrous iron [ 38 ], lipid peroxidation products may have contributed to the oxidation/reduction potential of the reaction. Conclusions Long-term supplementation studies where diet is controlled will probably be necessary to identify the benefits provided by lycopene. There may be real health benefits associated with lycopene especially since it is stored in various tissues and exhibits strong antioxidant activity in vitro [ 8 , 10 , 39 , 40 ]. Also the body of epidemiological evidence points to the protection provided against cardiovascular disease and some cancers with lycopene containing foods [ 11 , 12 , 41 , 42 ]. Recent cancer intervention studies have reported beneficial effects on prostate cancer from lycopene food supplementation [ 43 , 44 ]. The health benefits associated with diets providing lycopene are most likely long-term. Therefore, the findings of the present study should not be interpreted as a lack of health benefits from regular consumption of lycopene-rich foods. The interaction between cholesterol levels and antioxidant values needs more research. Contradictory findings of this study with other ex vivo antioxidant studies may be due to the cholesterol levels of subjects thus warranting further research. Competing interests None declared. Note Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. The article cited was prepared by a USDA employee as part of his/her official duties. Copyright protection under U.S. copyright law is not available for such works. Accordingly, there is no copyright to transfer. The fact that the private publication in which the article appears is itself copyrighted does not affect the material of the U.S. Government, which can be freely reproduced by the public. Authors' contributions JKC: conception and design of the study, drafted the manuscript, BHA: design of study, editing, PLC:design of the study, statistical analysis, PPV: conception of study, editing, RAB: editing, technical assistance, BAC:conception of study, editing. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521493.xml |
497047 | An active electrode for biopotential recording from small localized bio-sources | Background Laser bio-stimulation is a well-established procedure in Medical Acupuncture. Nevertheless there is still a confusion as to whether it works or the effect is just placebo. Although a plethora of scientific papers published, showing positive clinical results, there is still a lack of objective scientific proofs about the bio-stimulation effect of lasers used in Acupuncture. The objective of this work was to design and build a body surface electrode and an amplifier for biopotential recording from acupuncture points, considered here as small localized bio-sources (SLB). The design is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the bio-stimulation effect of lasers used in Medical Acupuncture. Methods The active electrode presented features a new adjustable anchoring system and fractionation of the biopotential amplifier between the electrode and the cabinet's location. The new adjustable electrode anchoring system is designed to reduce the electrode-skin contact impedance, its variation and motion artifacts. That is achieved by increasing the electrode-skin tension and decreasing its relative movement. Additionally the sensing element provides local constant skin stretching thus eliminating the contribution of the skin potential artifact. The electrode is attached to the skin by a double-sided adhesive pad, where the sensing element is a stainless steel, 4 mm in diameter. The fractionation of the biopotential amplifier is done by incorporating the amplifier's front-end op-amps at the electrodes, thus avoiding the use of extra buffers. The biopotential amplifier features two selectable modes of operation: semi-AC-mode with a -3 dB bandwidth of 0.32–1000 Hz and AC-mode with a bandwidth of 0.16–1000 Hz. Results The average measured DC electrode-skin contact impedance of the proposed electrode was 450 kΩ, with electrode tension of 0.3 kg/cm 2 on an unprepared skin of the inner forearm. The peak-to-peak noise voltage measured at the amplifier output, with input terminals connected to common, was 10 mV p-p , or 2 μV p-p referred to the input. The common-mode rejection ratio of the amplifier was 96 dB at 50 Hz, measured with imbalanced electrodes' impedances. The prototype was also tested practically and sample records were obtained after a low intensity SLB laser stimulation. All measurements showed almost a complete absence of 50 Hz interference, although no electrolyte gel or skin preparation was applied. Conclusion The results showed that the new active electrode presented significantly reduced the electrode-skin impedance, its variation and motion artifact influences. This allowed SLB signals with relatively high quality to be recorded without skin preparation. The design offers low noise and major reduction in parts, size and power consumption. The active electrode specifications were found to be better or at least comparable to those of other existing designs. | Background The non-invasive nature of laser bio-stimulation have made lasers an attractive alternative in Medical Acupuncture at the last 25 years. Unfortunately there is still a confusion as to whether they work or their effect is just placebo. Although a plethora of scientific papers published, showing positive clinical results, there is still a lack of objective scientific proofs about the bio-stimulation effect of lasers used in Acupuncture. The objective of this work was to design and built a body surface electrode and an amplifier for biopotential recording from acupuncture points. The latter are considered here as small localized bio-sources (SLB). As discussed by other authors, SLB are small area body regions with specific electrical, physiological and anatomical properties (e.g. high density of gap junctions, relatively low impedance etc.) [ 1 - 4 ]. They appear to be highly sensitive to mechanical, thermal, electrical or electromagnetic stimulation and are found to take place from the epidermis (stratum granulosum) to a maximum depth of 2 cm [ 5 - 8 ]. The active electrode is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the bio-stimulation effect of lasers used in Medical Acupuncture. Methods Electrode design The attempt to define the optimal parameters of the active electrode was based on a set of preliminary measurements performed in our laboratory. Anatomical, physiological and electrical characteristics of the signal source were considered. The SLB AC signal level, after stimulation, varied from subject to subject, but did not exceed 1 mV peak-to-peak (p-p). Additionally SLB occasionally manifested a high DC potential up to 200 mV, implying the use of differential amplifier with optional DC coupling and wide DC input range. The frequency band of the signal of interest was found to be in the range 0–200 Hz. The preliminary experiments showed that SLB potentials were best recorded with small pasteless electrodes although their contact impedance depends strongly on sweat gland secretion. The application of electrolytic gel resulted in significant reduction of the SLB signal amplitude, probably due to smoothing of the potential caused by saturation of the epidermis with electrolyte. Moreover, potentials between closely spaced SLB might be shortened by the application of excessive gel or large surface electrodes. An additional difficulty is that the SLB are often situated at convex or concave body surface areas where large flat electrodes could not be easily affixed. Skin abrasion with sandpaper is also not recommended since it can cause skin irritation and SLB potential changes. However, the use of small passive dry electrodes on an unprepared skin results in high electrode-skin impedance, motion artifacts, high power-line cable interference and noise. When the electrodes are DC coupled to the amplifier, a motion induced interfering signal appears at the amplifier input, mainly due to: • Electrokinetic effect – the disturbance of the double layer of charge at the electrode-skin interface causes variations of the DC polarization potential [ 9 ]. • Skin potential or skin stretch artifact – stretching of the skin causes a change of the potential of the barrier layer between the epidermis and the dermis [ 10 ]. • Variation in the electrode-skin contact resistance – caused by the amplifier input bias current and the current flowing due to the polarization potential. The complex electrochemical interactions that take place at the electrode-skin interface have been subject to much study in order an equivalent electrical model to be developed [ 10 - 12 ]. The simple but adequate electrical model used, is shown in Fig. 1 , where C d //R d is the coupling impedance of the double layer at the electrode-skin interface, C i //R i is the amplifier input impedance, R s is the minimum series contact resistance and V pol is the DC polarization potential. Then the motion artifact signal at the amplifier input can be expressed as Figure 1 Equivalent electrical model of the electrode-skin interface. V mot = Δ V pol + Δ V skin + (Δ R d + Δ R s ) ( V POL / R i + i b ) (1) where ΔV skin is the skin stretch artifact and i b is the amplifier input bias current. It was deduced that in order to keep the resistive interfering component less than 10 μV when DC coupling is employed and with both currents contributing equally to it, then i b <50 pA and R i >1 GΩ [ 11 ]. If an AC coupling is used then the resistive component of the motion artifact is eliminated. For dry electrodes the motion artifacts are mainly caused by changes of the polarization potential and the contact impedance due to the poor electrolyte layer at the electrode-skin interface. Therefore a firm electrode-skin contact is of primary importance. Thus a new adjustable electrode anchoring system was designed for the purpose, as shown in Fig. 2 . Turning the electrode cap clockwise pushes the sensing element against the skin. Thus electrode-skin pressure is increased, leading to reduction of the contact impedance and its variation. The electrode-skin relative movement is also reduced, making the noise contribution of the electrokinetic effect insignificant. Additionally, the sensing element provides constant skin stretching that lowers the contribution of the skin potential artifact. Turning the electrode cap counter-clockwise, releases the spring, which pushes back the sensing element, resulting in a lower electrode-skin pressure. Titanium, stainless steel and aluminum were considered as electrode sensing materials. Stainless steel was chosen because it is more commonly available than titanium and preferable to aluminum, which has been shown to have problems due to chemical response of its oxide to perspiration [ 13 ]. The use of stainless steel electrode material ensures low noise, minimal offset potentials and excellent DC stability suitable for low-drift DC measurements [ 14 ]. The sensing element is a 4 mm diameter, selected according to the average SLB size. Although the obtained contact impedances with the prototype electrode were relatively low for dry electrodes of that size, impedance matching at the electrode site was still needed to cope with the power-line cable interference. Figure 2 Dry active electrode with adjustable anchoring system. Basic amplifier circuit Electrodes with impedance matching at the sensing site are referred to as active electrodes and have been designed since 1960's [ 15 - 17 ]. The electronic part of these transducers mostly consists of a buffer amplifier, but some have been designed to need only two lead connection wire [ 18 , 19 ]. However, as the signal is not amplified, buffers introduce significant noise and a low noise amplifier is still needed at the front-end. In order to avoid this drawback we used a two-op-amp biopotential amplifier [ 20 ] shown in Fig. 3 , where op-amps A 0 and A 1 were integrated at the electrodes (Fig. 4 ), instead of using extra buffers. This resulted in lower noise and less parts, at the expense of increased number of electrode leads. The amplifier is based on the two-op-amp instrumentation amplifier shown in Fig. 5 . The output voltage of the basic two-op-amp amplifier is Figure 3 Schematic of the biopotential amplifier with active DC rejection/suppression. Figure 4 Simplified schematic of the biopotential amplifier with active electrodes. Figure 5 Schematic of the basic two-op-amp instrumentation amplifier. where A d1 (s) is the differential-mode (DM) gain and A c1 (s) is the common-mode (CM) gain of op-amp A 1 . If we take the usual definitions for the DM input signal, V d =(E 1 -E 0 ), and for the CM input signal, V c =(E 1 + E 0 )/2, then the output voltage can be also written as U 1 ( s ) = A d ( s ) V d + A c ( s ) V c (3) It can be shown that the respective expressions for the DM gain A d (s), and the CM gain A c (s), are given by where , τ 0 and τ 1 are the respective DM open loop gains, and the time constants of the first poles of op-amps A 0 and A 1 (assumed to be internally compensated), and A c1 (s) is the CM gain of op-amp A 1 . It should be noted that the CM gain of op-amp A 0 is omitted from (4) and (5), since its influence on both gains is insignificant. Considering (4) and (5), then the common-mode rejection ratio CMRR(s) is Assuming op-amps A 0 and A 1 are ideal then the only factor contributing to the CMRR is the mismatching of the resistors. Thus we can define a common-mode rejection ratio for the resistors, CMRR R . By taking 1/A d0 (s) = A c (s) = 0 in (6) we obtain Therefore CMRR R (s) approaches infinity if the relevant impedances are chosen according to If the condition in (8) is fulfilled and op-amps A 0 and A 1 are ideal, then (4) simplifies to Considering (7), equation (6) can be written as where CMRR A1 (s) is the CMRR of op-amp A 1 . Further if we assume that Z 1 , R 2 , R 3 and Z E have tolerance t, then from (7) and (8) we can deduce that the worst case condition will be when Z 1 = Z 10 (1-t), R 2 = R 20 (1+t), R 3 = R 30 (1-t) and Z E = Z E0 (1+t) where Z 10 , R 20 , R 30 , and Z E0 , are the respective nominal values. Equation (7) then can be written as where . This means that large differential gain is desirable since very small tolerance components are expensive. Therefore, considering (11), equation (10) can be written as The CMRR A1 has the form where ω r is the frequency where CMRR A1 has decreased by 3 dB and is usually between 100 Hz and 1 kHz. The open loop gain A d0 (s), also decreases at higher frequencies with a corner frequency ω 0 = 1/τ 0 which is usually lower than ω r , if A 0 and A 1 are of the same type. Therefore the CMRR is mainly determent at low frequency by the matching of the resistors and the DM gain, and at high frequencies by the open loop response of op-amp A 0 , rather than its CMRR. If we take the advantage of the fact implicit in (10), and achieve then theoretically the CMRR becomes infinite. In part this can be achieved by the use of a capacitor and resistor in parallel for the impedance Z 1 (Fig. 3 ), and then trimming R 2 . Thus the need of low-tolerance components is eliminated. Therefore Z 1 (s) will have the form It has been shown [ 20 ] that a good approximation for the optimal value of the capacitor C 1 is where GBP A0 is the gain bandwidth product of op-am A 0 . Trimming R 2 is a good solution for achieving an ultra high CMRR for demanding application, however it is not practical since the trimmer has to be incorporated in the electrode. Alternatively, Z E or R 3 can be trimmed, which however will alter also the amplifier DM gain. Considering equation (12) it can be shown that for application with relatively high DM gain and proper op-amp selection, both trimming and compensation (C 1 ) can be omitted, without significantly degrading the CMRR. For example, if the usual 1% tolerance resistors are used and op-amps with CMRR of 100 dB and DM open loop gain of 120 dB at 50 Hz, then for an amplifier with DM gain of 5000, a CMRR of 96 dB can be achieved without trimming. In the amplifier circuit shown in Fig. 3 , Z E is replaced with an active DC rejection/suppression circuit [ 20 ]. It includes an integrator (A 2 , R i , C i ) and two potential dividers (R 6 , R 5 and R 4 , R 3 ). The amplifier can operate in AC-mode or in semi-AC-mode. The two modes are selectable by the switch S 1 : AC-mode with S 1 open and semi-AC-mode with S 1 closed. In AC-mode the DC signals are rejected, where in semi-AC-mode they are suppressed. If R 6 = R 6 *, R 5 = R 5 * and R i = R i *, then the respective expressions for the equivalent impedance Z E (s) for the two modes are given by where τ i = R i C i is the time constant of the integrator, and τ 2 are the respectively the DM open loop gain and the time constant of the first pole of op-amp A 2 . Whenever R i >>R 5 then k ≈ (R 6 /R 5 +1), which is true with the time constants and voltage gains, typical in biopotential recordings. For signals bellow the amplifier high-pass cut-off frequency, Z E (s) decreases due to the active DC rejection/suppression circuit. For DC signals equation (8) is maximally imbalanced and thus CMRR R (0) ≈ A d (0). Since for biopotential amplifiers A d (0) is much lower than CMRR A1 (0) and A d0 (0), therefore CMRR(0) ≈ A d (0), which represents the worst case. If we consider only the -3 dB bandwidth and assume that op-amp A 2 is ideal, then (17) and (18) simplify to Therefore, in this case (9) can be written as which represents the mid band DM gain for both modes. After substituting (17) and (18) in (9), it can be shown that the respective DC differential gains for the two modes are given by where 2 k is approximately the DC gain of the stopped integrator (A 2 , R i , C i , R i *, R 5 *, R 6 *) in semi-AC-mode. Thus DC signals meet lower gain, in order to prevent saturation from large electrode offsets or other high DC potentials. The active electrodes' input resistances R iE0 and R iE1 , are not equal due to the different closed loop gains of op-amps A 0 and A 1 , and can be expressed as where R iA0 and R iA1 are the input resistances of op-amps A 0 and A 1 . However, at higher frequencies, the electrodes' input impedances are much lower and about the same (assumed that A 0 and A 1 are of the same type), due to the op-amps' input and additional stray capacitance, being in parallel to the high op-amps' input resistance. The output noise spectral density for the -3 dB bandwidth is approximately the same for both modes and can be written as where e n0 , e n1 and e n2 are the respective voltage noises of op-amps A 0 , A 1 and A 2 . Assuming E 0 is connected to common (Fig. 3 ), then the amplifier transfer function H(s) is given by After substituting Z E (s) and A d1 (s) in (24), it can be shown that H(s) has three poles and two zeros for both modes. However, with the time constants and voltage gain used in the current application, one pole almost coincides with one zero. Therefore, H(s) can be approximated very well by a transfer function with two poles and one zero. The respective approximations for AC-mode and semi-AC-mode are given by The circuits described by the transfer functions H AC (s) and H sAC (s) are stable because all the poles are situated in the left half of the complex s-plane and there are no resonance effects as the poles are on the real s-axis. Practical amplifier circuit The schematic of a multichannel amplifier with active electrodes, built according to the design discussed is shown in Fig. 6 . Each channel amplifies the signal between its input (E 1 ...E N ) and the reference input E 0 (monopolar configuration). The output voltage of op-amp A 2 is equal to the DC input voltage, multiplied by the ratio (R 3 + R 4 )/R 3 for both modes. The choice of the resistor ratio (R 3 + R 4 )/R 3 is a trade-off between DC input range and noise, since a low ratio enhances the noise contribution of op-amp A 2 . The ratio R 4 /R 3 was chosen so that to allow a DC input voltage range of ± 370 mV, without saturating op-amp A 2 . The offset voltage at the amplifier output (U1) is the input offset voltage of op-amp A 2 , times the resistor ratio (R 5 + R 6 )/R 5 . In case of high DM gain, the output offset voltage would become unacceptably high. Thus, op-amp A 2 was selected for its ultra-low offset voltage of 1 μV, low noise and high CM input range to prevent latch-up. Moreover A 2 is a low input bias current type, which allows the use of high value input resistances without producing large offset voltages between its inputs. Op-amp A 0 , was selected with a relatively high GBP, in order to confine its influence on the CMRR at higher frequencies. Combining high DM gain, with high GBP op-amps, allowed Z 1 to be implemented only with its active part R 1 , as thus the CMRR at 50 Hz was not significantly degraded. As shown in (23), the equivalent input noise is mainly determent by the noise of op-amps A 0 and A 1. They were implemented with the low-noise CMOS op-amp LMV751 in a SOT23-5 package. Figure 6 Multichannel biopotential amplifier with active electrodes and DRL circuit. Because of the large integrator's time constant, the amplifier has a very slow response after overloads (≤ 10τ i ), caused by large signal disturbances. Thus a deblocking circuit was added at the cabinet's location, for temporary reduction of the time constant during overload [ 24 ]. It is controlled by the output voltage U1 through the low pass filter (R 13 , C 2 ). The filter output controls two threshold triggers (A 3 , A 4 ), which through D 1 ,D 2 control the MOS transistor T 1 , acting like a switch. When the output signal reaches its range limits (defined by R 14 , R 15 , R 16 ), T 1 opens and the new reduced time constant τ i * = (R 7 //R 11 )C 2 , pulls the output signal to the zero level. This state is maintained for additional hundred milliseconds (R 13 C 2 ) and then is switched back to its original value. The connection between the amplifier common and the signal source is implemented by a driven right-leg (DRL) circuit. The CM voltage at the output of A 0 is reduced by a factor equal to the DRL circuit gain (A DRL = 314 at 50 Hz), which theoretically should give a 50 dB extra CMRR at 50 Hz. In addition, in case of a faulty op-amp, the DRL circuit will limit the maximum patient current to a safe level of 50 μA. Results The contact impedance of the proposed electrode, measured and averaged over five subjects, and its calculated model impedance are shown in Fig. 7 . The values of the model elements were determent to give the closest agreement between the measured electrode-skin impedance and that of the model. The values are: R s = 300 Ω, R d = 450 kΩ and C d = 3 nF. The measuring technique is described by Bergey et al. [ 21 ] and was performed five minutes after the application of the electrodes to allow their impedance to settle to a constant value [ 13 ]. The electrodes were applied with moderate tension of 0.3 kg/cm 2 on an unprepared skin of the inner forearm. The impedance was lower when higher tension was applied or when sweat was present on the skin. The applied current density was 0.01 mA/cm 2 and no current density impedance dependence was observed [ 22 ]. The electrode-skin impedance showed two-decade spread between different subjects, which was also reported in other works [ 23 ]. Figure 7 The average electrode-skin contact and the calculated model impedance against frequency. The plot shows also the minimum and the maximum data sets for five subjects. Simulations of the amplifier circuit were carried out using PSPICE. The op-amps used in the model were with gain-bandwidth product (GBP) of 5 MHz, DM open loop gain of 120 dB and CMRR of 100 dB. The integrator time constant and the resistor ratios of the feedback loop were: τ i = R i C i = 1, R 4 /R 3 = 6 and R 6 /R 5 = 700. The amplifier frequency response plots, for both operating modes, are shown in Fig. 8 . In semi-AC-mode the high-pass response is with 1 st order pole at 0.32 Hz and zero at 0.2 mHz. In AC-mode the high-pass response is with 1 st order pole at 0.16 Hz and zero at 0.16 μHz. The mid band DM gain is the same for both modes. Simulations were also carried out for the estimation of the power line interference due to induced displacement currents into the electrode leads. The increased stray capacitance between the power line and the amplifier, caused by the increased number of electrode leads, was taken into consideration in the simulation model. The results showed that the interference caused by the active electrode unshielded leads was insignificant. Figure 8 A plot of the amplifier frequency response. Table 1 shows the amplifier specifications, measured with battery powered prototype and test equipment. All the parameters were in close agreement with those of the simulations. The peak-to-peak noise voltage measured at the amplifier output, with input terminals connected to common, was 10 mV p-p , or 2 μV p-p when referred to the input. The CMRR of the amplifier was 96 dB at 50 Hz, measured with imbalanced electrode impedances (ΔZ e = 47 kΩ). The maximum measured CMRR with DRL and a CM input signal of 4 V p-p , was 126 dB at 50 Hz, where the output signal level was approximately equal to the amplifier output voltage noise. Table 1 Active electrode specifications Parameter semi-AC-mode AC-mode Bandwidth (-3 dB) 0.32–1000 Hz 0.16–1000 Hz DC gain 3.22 ≈ 0 AC mid band gain 74 dB Differential mode AC input range 0.005–1 mV p-p Differential mode DC input range ± 370 mV Common mode input range ± 2 V Input noise current 1 pA rms @ 0.1–200 Hz Input bias current 1.5 pA Input impedance, Active Electrode 320 MΩ @ 50 Hz (1000 GΩ //10 pF) CMRR 96 dB @ 50 Hz Output offset 0.7 mV Input noise voltage 2 μV p-p (0.33 μV rms ) @ 0.1–200 Hz Power consumption 11 mW @ one channel A sample record from the practical application of the active electrode, obtained after a low intensity SLB laser stimulation, is shown in Fig. 9 . The amplifier was battery powered and optically isolated by linear optocouplers. The bandwidth was limited to 200 Hz, by 6 th order low pass Bessel filter, and the signal was sampled with 1 kHz. The electrodes were connected with a high-density unshielded ribbon cable. One electrode (E 1 ) was placed on SLB TH-23, near the eyebrow, where the reference (E 0 ) was placed on the ear lobe. No electrolyte gel or skin preparation was applied. The measurements were performed in a typical laboratory room. All measurements showed almost complete absence of 50 Hz interference. The noise present in the signal is mainly compounded of artifacts from eye movements, electromyographic signals, and noise from the electrode-skin interface. Figure 9 Biopotential acquired in semi-AC-mode from SLB TH-23 after low intensity laser stimulation. Discussion The best solution for an active electrode would be to perform the entire analog signal processing at the electrode site. This could be achieved with a custom made integrated circuit, but the cost would be much higher. We found a good alternative in using SMD technology and integrating only the front-end of the amplifier into the electrode. The ultra high input resistance of the electrode is degraded at higher frequencies by the op-amp's input capacitance in parallel with the stray capacitance due to the electrode Printed Circuit Board (PCB). Nevertheless, combining an op-amp with low input capacitance and a proper PCB design, allowed a relatively high input impedance to be achieved at 50 Hz. That decreased the amplifier sensitivity to high electrode-skin impedance imbalances, by reducing the transformation of the CM interference signal into unwanted DM signal. Unfortunately, most data sheets do not properly specify op-amp's input capacitance, neither DM nor CM. The active electrode presented is not suitable for applications requiring a low differential gain and large signal bandwidth due to the decreasing CMRR at higher frequencies, if not properly compensated. On the other hand, below the high-pass cut-off frequency, the CMRR is degraded by the active feedback circuit, and reaches its minimum value for DC signals, equal to the DM gain. The circuit can accept high value input filter resistances, which will also limit the patient auxiliary current in case of fault condition of op-amps A 0 and A 1 . Because of the limited electrode space, it is preferable that the front-end op-amps feature internal electrostatic discharge protection circuitry, rather than building an external one. Conclusions The new electrode anchoring system significantly reduced the electrode-skin impedance, its variation and motion artifact influences. The proposed amplifier fractionation resulted in lower noise and less parts. Moreover splitting the amplifier between the electrodes and the cabinet's location allowed the use of an automatic DC deblocking system and mode switching. The prototype tests showed that with the active electrode presented, SLB signals with relatively high quality could be recorded without skin preparation. The 50 Hz interference pickup by the electrode leads was practically eliminated. Because high electrode-skin impedances are tolerated, no electrolytic gel is needed. This allows fast application of the electrodes, minimizes patient discomfort and eliminates the risk of infection. With proper op-amps selection, the active electrode specifications were found to be better or at least comparable to those of other existing designs. The design offers low noise and major reduction in parts, size and power consumption. It is currently used in studying laser provoked SLB potentials and their propagation, aiming to gain a better insight into the bio-stimulation effect of lasers in Medical Acupuncture. Authors' contributions The authors contributed equally to this work | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC497047.xml |
554756 | The Future of Smallpox Vaccination: is MVA the key? | Eradication of the smallpox virus through extensive global vaccination efforts has resulted in one of the most important breakthroughs in medical history, saving countless lives from the severe morbidity and mortality that is associated with this disease. Although smallpox is now extinct in nature, laboratory stocks of this virus still remain and the subject of smallpox vaccination has gained renewed attention due to the potential risk that smallpox may be used as a biological weapon by terrorists or rogue states. Despite having the longest history of any modern vaccine, there is still much to be learned about smallpox vaccination and the correlates of protection remain to be formally defined. This Commentary will discuss the strengths and weaknesses of traditional smallpox vaccination in comparison with immunization using modified vaccinia virus Ankura (MVA), a non-replicating virus with a strong safety record but weakened immunogenicity. | Introduction Smallpox (Variola major) is a virus that no longer exists in the wild, but during its reign it caused 20–30% mortality in previously unvaccinated individuals and often left survivors with deeply pitted scars for life [ 1 ]. The last case of smallpox in the U.S. occurred in 1949 and the last case of naturally occurring smallpox in the world occurred in Somalia in 1977. Smallpox has no known animal reservoir, so in the absence of any more natural cases of human smallpox being recorded after 1977, the virus was considered fully eradicated in 1980 [ 1 ]. Despite extinction in nature, smallpox virus stocks still reside in secure locations within the U.S. and Russia but it is impossible to know if other undeclared stocks of smallpox remain in other countries [ 2 ]. Moreover, in the age of genetic engineering it is possible that more virulent strains of smallpox or other potentially dangerous orthopoxviruses could be developed and unleashed in an effort of bioterrorism. Although the potential for developing a pathogen more lethal than wild smallpox is theoretically possible [ 2 , 3 ] it would by no means be a simple task to undertake and the outcome would likewise be uncertain [ 4 , 5 ]. Nevertheless, smallpox is considered a potential risk to national security and efforts are underway to prepare the United States and several other countries for a deliberate release of smallpox as a biological weapon. The first line of defense against smallpox is vaccination. Smallpox vaccination is highly effective at protecting against lethal infection and even if only partial protective immunity is attained, this often still results in survival and decreased viral spread to others (Chapter 4, pages 189–90 of [ 1 ]). The smallpox vaccine was discovered by Edward Jenner, who was the first to prove that infection by cowpox resulted in protective cross-reactive immunity against smallpox [ 6 ]. Dr. Jenner not only demonstrated that cowpox could induce protective immunity, but in answer to critics of his day who argued that this form of immunity would only be short-lived, he demonstrated full protection against smallpox in several individuals at 25, 27, 31, 38, and even 53 years after cowpox infection [ 6 , 7 ]. It is not a coincidence that such long-term time points were examined. As Dr. Jenner noted, "I have purposely selected several cases in which the disease [i.e. cowpox] had appeared at a very distant period previous to the experiments made with variolous matter, to show that the change produced in the constitution is not affected by time." [ 6 ]. Following the elegant studies initiated by Edward Jenner, the world was eventually freed of the scourge of smallpox following a massive global eradication campaign [ 1 ] and although hugely successful, there were still many questions that were left unanswered. These questions represent the topics of this commentary. For instance, by then end of the 1960's it was realized that smallpox vaccination was the cause of a substantial number of adverse events and resulted in a lethal infection in approximately one out of one million people who received this live viral vaccine. In Germany, an extremely safe attenuated strain of vaccinia, known as modified vaccinia virus, Ankura (MVA) was developed [ 8 , 9 ], but it was only used as primary vaccination followed by traditional smallpox vaccination. Moreover, its efficacy against smallpox was never directly tested due to the eradication of smallpox shortly thereafter and the question remains as to whether it would induce full or only partial immunity when faced against fully virulent smallpox. Although the virology, pathology, and epidemiology of smallpox are well described [ 1 ], there is a relative dearth of information regarding the immunology of smallpox and smallpox vaccination. Most importantly, there is currently no consensus on the immunological correlates of protection, making it difficult to implement rationale vaccine design when it not established which immunological benchmarks are necessary for full or even partial protection. Recent quantitative analysis of the cellular and humoral immune response following smallpox vaccination, coupled with historical evidence of protective immunity, are beginning to shed light on this important subject. Discussion The question of safety following smallpox vaccination The current smallpox vaccine was prepared prior to 1982 under standards that today, would be unlikely to be approved by the FDA. To produce the vaccine, the torso of bovine calves were shaved, and their skin scarified (scratched) with an inoculum containing vaccinia virus. After the infection has reached a point in which a great deal of exudate was observed, the purulent lymph material was scraped from the infected cow, clarified, and lyophilized in the presence of antibiotics. The inoculum is later reconstituted with diluent containing 0.25% phenol to further decrease bacterial contamination (<200 viable bacterial/mL after reconstitution; see Dryvax package insert). Unlike most other vaccines that are administered by subcutaneous or intramuscular injection, vaccinia virus replicates poorly under these conditions and optimal vaccination occurs by scarification of the virus inoculum onto the skin surface [ 10 ]. Viral replication on the skin surface typically results in a vesicular or pustular lesion that is described as a "take" which later crusts over and sloughs off, leaving behind a small scar. Since the time of Edward Jenner, the presence of a vesicular or pustular lesion has remained the gold-standard measurement of successful vaccination. The success of smallpox vaccination does not come without unwanted consequences. The most common side effects of smallpox vaccination are fever and other flu-like symptoms. More serious adverse events include inadvertent inoculation (529 cases/10 6 doses), generalized vaccinia (242 cases/10 6 doses), eczema vaccinatum (39 cases/10 6 doses), vaccinia necrosum (1.5 cases/10 6 doses), encephalitis (12 cases/10 6 doses), or death (~1 death/10 6 doses) [ 11 ]. The mortality rate is highest for infants that are <1 year of age (5 deaths/10 6 doses) whereas the mortality rate for older children and adolescents aged 1–4 or 5–19 is approximately 0.5 deaths/10 6 doses (see also [ 12 , 13 ] and Dryvax package insert). It is rare for adults to die after smallpox vaccination; of 68 deaths attributed to smallpox vaccination over a 9-year period of evaluation, only 8/68 (12%) of cases occurred in adults. Five of the eight adults were over the age of 60 and 4/8 of the lethal cases occurred in adults who were diagnosed with terminal cancer at the time of vaccination [ 11 ]. Myopericarditis is a recently identified adverse event that occurs at a rate of ~124 cases/10 6 smallpox vaccinations [ 14 , 15 ]. In 2003, there were three fatal heart attacks that were temporally related to smallpox vaccination and this triggered critical evaluation of any vaccine-related cardiac events thereafter. It was later realized that all three heart attack victims (age 55, 55, and 57) had pre-existing risk factors for cardiac disease including hypertension, hyperlipidemia, and smoking. At autopsy, none of the three heart attack victims showed signs of myo/pericarditis but the deaths were instead linked directly to ischemic events [ 16 ]. A retrospective study analyzing the number of cardiac deaths among approximately 80,000 death certificates issued near the time of a massive smallpox vaccination effort in New York City in 1947 has also brought new insight into the dangers of smallpox vaccine-induced myocarditis[ 16 ]. Following an imported case of smallpox, about 6.4 million people of all ages were vaccinated in a one-month period. Analysis of the frequency of cardiac deaths before, during, and after the vaccination campaign failed to show a statistical increase in these events. Moreover, analysis of 64 recently identified cases of myocarditis in the U.S. military smallpox vaccination program found that approximately 80% of patients reported no long term sequelae and 100% of patients demonstrated objective normalization of echocardiography, electrocardiography, laboratory testing, graded exercise testing, and functional status [ 15 ]. To overcome some of the problems associated with the current calf lymph smallpox vaccine (Dryvax), an improved tissue culture-derived vaccine has been recently developed [ 17 ]. Unlike Dryvax, this new vaccine (designated ACAM1000) is produced under sterile GMP conditions, so bacterial contamination is avoided. Also, unlike Dryvax which contains a heterologous mixture of virus variants with some differing in their neurovirulence [ 17 , 18 ], ACAM1000 is clonally derived (triple plaque-purified) and tested extensively for low neurovirulence in animal studies. Thus, it is hopeful that ACAM1000 vaccine will have reduced risk of encephalitis, one of the major risk factors following smallpox vaccination. It remains to be seen whether or not myopericarditis or other potentially serious adverse events will be affected by the use of this new vaccine. Both Dryvax and ACAM1000 represent replication-competent smallpox vaccines and since these vaccines are based on the use of live viruses, there is always an inherent risk of severe adverse events or death (albeit rare) in vaccinees that have unknown or undisclosed immunodeficiencies at the time of vaccination. To overcome the hazards of replicating viruses, a highly attenuated strain of vaccinia, designated Modified Vaccinia virus Ankara (MVA) was developed by growing the virus for >500 passages on chicken embryo fibroblasts (CEF) and following the loss of about 15% of its parental genome, it no longer was capable of replicating in most mammalian cells, including human cells [ 19 ]. Other strains of non-replicating orthopoxviruses have also been developed, including NYVAC, which was derived from vaccinia virus following a deletion of 18 genes – including those encoding virulence factors and human host range replication, and ALVAC, an attenuated viral vector that is based on canarypox, an avipoxvirus that grows only in avian species. In one study, recombinant NYVAC and recombinant ALVAC expressing JEV proteins were found to be well tolerated but more reactogenic than the commercially available formalin-inactivated JEV vaccine [ 20 ]. Of these attenuated poxvirus vaccine strains, MVA is the one with the most extensive history of safety in humans. Beginning in 1968, >100,000 people in Germany were vaccinated with MVA (followed by traditional smallpox vaccination) and although it was well tolerated, MVA was not used alone and since there were no smallpox outbreaks at that time, its efficacy in the face of an actual smallpox outbreak has not been tested. With an excellent safety profile in humans and in animal models of immunodeficiency, recombinant MVA expressing candidate immunogens from a variety of infectious agents (e.g. HIV, HPV, and malaria) or tumor antigens (e.g. melanoma) have now reached Phase I and Phase II clinical trials [ 21 , 22 ]. However, the role of MVA in the future of smallpox vaccination has yet to be decided and will likely be determined by the outcome of clinical trials that directly compare the immunogenicity of MVA to either Dryvax or ACAM1000, two vaccines that are likely to provide the required high levels of protective immunity that will be necessary in the event of an accidental or deliberate smallpox outbreak. Quantitative analysis of vaccine efficacy Edward Jenner developed the first test for vaccine efficacy when he immunized subjects with cowpox and then later challenged them with smallpox by inoculation. If a subject showed no secondary smallpox lesions indicative of systemic spread, then the individual was believed to have protective immunity. He noted that cowpox-vaccinated individuals who developed a pox-like lesion at the vaccination site were fully protected against smallpox challenge. To this day, most studies still use the identification of a "take" (vesicular or pustular lesion) as evidence of successful vaccination. However, there are rare cases of revaccinated subjects who present with vesicle formation after smallpox vaccination, but show no detectably boosted cellular or humoral immunity [ 23 ] and M.K. Slifka, unpublished results). For this reason, it is important that pre- and post-vaccination serum antibody and peripheral T cell responses be monitored in individuals who plan to work with virulent orthopoxviruses or who would be expected to enter a hot zone in the case of a smallpox outbreak. Quantitative immunology is beginning to gain acceptance as a measurement of vaccine efficacy, although the Jennerian vesicle at the site of smallpox vaccination still remains the primary endpoint of successful vaccination in most studies [ 17 , 23 - 25 ]. One of the reasons why quantitative immunology is more important now than ever before is that with some vaccines such as MVA, there is no vesicle formed due to the route of immunization – so unless immunological measurements are made, then vaccine immunogenicity cannot be determined or compared. Humoral immunity following smallpox vaccination was measured in the 1960's and 1970's by means of neutralizing assays (primarily against the IMV form of vaccinia or smallpox) and today, humoral immune responses are quantitated by analysis of neutralizing activity against IMV or EEV forms of vaccinia or by the use of ELISA assays using whole-virus lysate and/or individual vaccinia IMV or EEV proteins. Moreover, the vaccinia-specific memory B cell response has also been recently studied [ 26 ] providing the first direct quantitation of this memory cell subset. Quantitation of the antiviral T cell response mounted after smallpox vaccination was not an option during the smallpox era because the tools and technology were not available for analysis of cellular immunity. In contrast, today there are now several sophisticated techniques that can be used to monitor antiviral T cell responses directly ex vivo including vaccinia-specific IFNγ ELISPOT assays [ 17 , 23 , 26 - 28 ], intracellular cytokine staining analysis (ICCS) [ 29 - 33 ], or peptide/MHC Class I tetramer staining [ 28 , 30 ]. Each of these techniques has high sensitivity and high specificity and each has both advantages and disadvantages. For instance, the IFNγ ELISPOT assay provides a highly sensitive calculation of IFNγ-producing cells, but one drawback is that it only allows detection of one cytokine at a time and the phenotype of the IFNγ-producing subset (CD4, CD8 or possibly NK cells) must be determined by purifying each population independently prior to the assay. The advantage of ICCS is that antiviral T cells can be quantitated based on the production of more than one cytokine and the phenotype of the responding lymphocyte subset is directly determined by flow cytometry. The main disadvantage of ICCS is that a relatively large number of cells are required in order to detect rare populations of virus-specific T cells. The advantage of using peptide/MHC tetramers is that CD8 + T cells can be quantitated regardless of their cytokine profiles, but the main disadvantages with this approach include the lack of identified CD4 + T cell/MHC Class II epitopes, the requirement for knowledge of the MHC haplotype of the subject, and the inability to measure the total antiviral T cell response, which may be directed against any number of immunodominant and subdominant peptide epitopes. Direct quantitation of the antiviral immune response induced by smallpox vaccination has been critical for several recent advances in orthopoxvirus immunobiology – especially since smallpox has been eradicated and other human orthopoxvirus outbreaks are too small and sporadic (e.g. cowpox [ 34 , 35 ] or monkeypox [ 36 , 37 ]) to be feasible for field studies of protective efficacy. For example, Weltzin et al . [ 17 ] not only compared the neutralizing titers induced by Dryvax vs. ACAM1000 vaccines, but also compared vaccinia-specific T cell responses by IFNγ ELISPOT as well as by cytolytic T cell assays and proliferation assays that, although less quantitative than the ELISPOT assay, are nevertheless important for analysis of antiviral T cell functions. These techniques allowed the investigators to demonstrate non-inferiority of the new tissue culture-derived smallpox vaccine compared to the Dryvax vaccine that is currently in use. Another study by Earl et al. [ 38 ], used quantitative immunology to compare vaccine efficacy of Dryvax, MVA followed by a Dryvax booster, and MVA followed by an MVA booster in a cynomolgus monkey ( Macaca fascicularis ) model of monkeypox infection. Using ELISA assays and neutralizing assays to measure vaccinia-specific antibody responses, and ICCS to measure antiviral T cell responses, the authors showed that antiviral immunity appeared similar between these three groups. Likewise, each of these groups were protected against lethal monkeypox challenge, although primates that only received MVA plus an MVA booster showed partial protection with 6/6 animals presenting with 1–36 monkeypox lesions (compared to >500 monkeypox lesions in the unvaccinated controls). This indicates that two MVA vaccinations are required to induce partial immunity whereas MVA followed by Dryvax immunization or a single Dryvax immunization each provides full immunity. The protective efficacy of a single dose of MVA is unknown, but based on the results of the Earl et al. study [ 38 ] wherein two doses were required to elicit partial immunity, it appears likely that a single dose of MVA would be of only low protective value in the face of a virulent orthopoxvirus infection. This is not surprising since MVA, NYVAC, and ALVAC (all replication-deficient vaccines), typically require booster doses to be administered in order to elicit optimal immune responses. Moreover, several prime-boost strategies (including DNA vaccination followed by MVA booster) are being tested in order to overcome the low immunogenicity of MVA alone [ 21 ]. This is different from most replicating viruses [ 39 ] including vaccinia, which require only one immunization (or infection) to induce optimal and often lifelong immunity [ 5 , 26 , 32 , 40 ]. Approximately half of the U.S. population has been vaccinated against smallpox and continue to maintain pre-existing antiviral immunity [ 26 , 32 ] and this may have an impact on the efficacy of MVA vaccination. For instance, studies involving the closely related, non-replicating NYVAC strain have found that pre-existing immunity significantly effected the outcome of vaccination [ 20 ]. In this particular study, immunization with recombinant NYVAC expressing JEV proteins failed to induce protective antibodies in 0/5 vaccinia pre-immune individuals and although 5/5 vaccinia-naive subjects seroconverted after NYVAC-JEV immunization, the resulting neutralizing titers were substantially lower than that observed in subjects who received the standard formalin-inactivated JEV vaccine [ 20 ]. Oddly, the authors noted that both the NYVAC-JEV and the ALVAC-JEV vaccines (2 doses administered 28 days apart) failed to induce a detectable anti-vaccinia neutralizing response. It is difficult to speculate the efficacy of MVA vaccination of humans as a method of protection against smallpox. It is possible that booster doses of MVA would provide strong enough immunity to at least protect against lethal smallpox, similar to its ability to protect primates from lethal monkeypox [ 38 ]. On the other hand, clinical trials could indicate that MVA vaccination alone may be too inconsistent or only induce low levels of immunity that would be considered inferior to live smallpox vaccination with calf-lymph or tissue culture-derived vaccine preparations. Unlike live smallpox vaccination, which can be used as an effective post-exposure treatment against the lethal consequences of smallpox [ 1 , 5 ], it is unlikely that the low immunogenicity of MVA would be capable of fulfilling this role. Moreover, in the event of a smallpox outbreak there would not be enough time to administer two or more doses of MVA if people were at a high risk of exposure. Under these circumstances, use of live viral vaccines would be critical for ring vaccination or mass vaccination scenarios. This is not to say that MVA would not have a potential role in biodefense strategies. For instance, in a pre-event scenario, one could foresee the use of MVA followed by vaccination with a live viral vaccine such as Dryvax or its equivalent. Under these circumstances, MVA would likely induce partial immunity that would reduce the adverse events that are associated with traditional smallpox vaccination. Moreover, MVA is the vaccine of choice in immunocompromised individuals with suppressed immune systems (cancer patients, organ-transplant patients, AIDS patients, etc.) who would otherwise be contra-indicated for administration of the live viral vaccines. More studies will be needed to determine the immunogenicity and protective efficacy of MVA and other related non-replicating vaccines in terms of their potential to counter a smallpox outbreak. The need for formal definition of protective immunity and the correlates of immunity There is more than one definition of protective immunity against smallpox. There is protection against infection, protection against disease, and protection against death. Of these, one might argue that protection against lethal infection is the ultimate definition of protective immunity. However, protection against infection and protection against disease not only reduce the morbidity of an outbreak, but these high levels of protective immunity are also associated with reduced virus spread to others (Chapter 4, pages 189–90 of [ 1 ]). Protection against infection is the most rare level of protective immunity since this requires that an infection be blocked at the point of entry. A meta-analysis of 10 epidemiological studies on smallpox noted that on average, the virus only infected ~4% of previously vaccinated household contacts (Chapter 4, pages 189–90 of [ 1 ]). However, these results were based on whether or not the vaccinated contacts showed disease symptoms and did not necessarily prove that they were never infected per se . Further analysis indicated that approximately 10% of previously vaccinated household contacts of smallpox patients were actually infected with smallpox as demonstrated by isolation of infectious virus from pharyngeal mucosa, but only 4/34 (12%) of these subjects developed the clinical symptoms of smallpox [ 41 ]. Moreover, another study showed that about 50% of previously vaccinated, disease-free contacts demonstrated serological results indicative of a recent orthopoxvirus infection [ 42 ]. This suggests that most instances of "protection against infection" may not be complete protection. Instead, many of the individuals thought to have had protection against infection may have actually been infected with smallpox but didn't know it because they were clinically asymptomatic. Similar to these historical studies, during the U.S. monkeypox outbreak in 2003 [ 37 ] we have identified three previously unreported cases of monkeypox in subjects who had received smallpox vaccination many years earlier and were unaware that they had become infected with monkeypox because they were spared any recognizable disease symptoms (M.K. Slifka, unpublished data). A major issue in the smallpox field is that there is no consensus on what is exactly required for protective immunity against this disease. In the age of quantitative immunology, we are beginning to find clues that might help answer this age-old question. In a study of >300 subjects, the levels of vaccinia-specific serum antibody and antiviral T cell responses were determined from 30 days to up to 75 years after smallpox vaccination [ 32 ]. Antiviral antibody responses were maintained essentially for life, whereas antiviral CD4 + and CD8 + T cell responses declined with a half-life of approximately 8–15 years, with CD4 + T cell memory being more stable than CD8 + T cell responses. Similar duration of antibody production was demonstrated by other recent studies as well as older literature [ 24 , 26 , 40 ]. Moreover, the gradual loss of T cell memory was also confirmed [ 26 ], as was the differential loss of CD8 + T cell memory over CD4 + T cell memory [ 33 ]. Based on historical analysis of vaccine-mediated protection against lethal smallpox (dating back to the age of Edward Jenner), indicates that protective immunity is often lifelong [ 40 ]. One might argue that if protective immunity against smallpox had an absolute requirement for antiviral CD4 + or CD8 + T cells, then protective immunity would not be life-long but would instead be more likely to decline at the same rates as T cell memory. This suggests that in humans, humoral immunity might play a more important role in protection against lethal infection than cellular immunity [ 5 , 40 ]. Smallpox disease symptoms become more pronounced with increased time since vaccination [ 43 ], and it likely that the combination of intact cellular and humoral immunity together provide the most robust antiviral immunity. Recent vaccinia studies in mice using either antibodies to deplete T cell subsets or mouse strains that are genetically deficient in CD4 + T cells or CD8 + T cells (or both) have indicated that, as long as there is strong humoral immunity against vaccinia, T cell memory is dispensable for protective immunity [ 44 - 46 ]. This result corresponds well with other studies in which protection against vaccinia or smallpox has been clearly demonstrated by the adoptive transfer of immune serum in humans or by transfer of monoclonal neutralizing antibodies in animal models [ 5 , 40 ]. On the other hand, adoptive transfer of virus-specific T cells and experiments in mice that are genetically deficient in B cells, also indicate that in the absence of pre-existing antibody responses, memory T cells can play an important role in protection. This indicates that cellular and humoral immunity have overlapping roles in protective immunity and one may compensate for a deficiency in the other. Based on the overlapping roles for T cell and B cell memory on protective immunity, is there any chance that a consensus can be reached in regard to defining an immunological correlate of immunity? One way to address this issue is to examine historical studies in which an immunological correlate was identified. In this regard, there are two independent studies in which investigators showed that subjects with vaccinia-specific neutralizing antibody titers of >1:20 [ 47 ] or ≥ 1:32 [ 48 ] were fully protected against smallpox. The latter study was the largest of the two and showed that 3/15 (20%) of subjects with titers below 1:32 contracted smallpox whereas 0/127 (<1%) of subjects with antibody titers of ≥ 1:32 contracted the disease. The only caveat to these studies is that the subjects also received post-exposure vaccination at the same time that serum samples were drawn, so the full protection afforded to the subjects with high pre-existing neutralizing titers may have been due to their high antibody titers, or the combination of strong pre-existing antibody titers in the context of post-exposure vaccination. A neutralizing titer of 1:32 is equivalent to a vaccinia ELISA titer of 944 Elisa Units (EU) or approximately 4 International Units (IU) of the WHO/NIBSC International Smallpox Serum Standard [ 32 ]. Interestingly, about 50% of subjects vaccinated in the distant past maintain neutralizing titers of 1:32 or greater for life and this coincidentally is the same proportion of vaccinated smallpox contacts who demonstrated fully protective immunity when exposed to smallpox-infected family members [ 42 ]. This leads one to speculate that 4 IU may constitute a protective level of serological immunity against smallpox. This is a testable hypothesis and to determine if this is a correlate of protection, it will be important to perform adoptive transfer of VIG or its equivalent into non-human primates (resulting in antiviral serum antibody levels of approximately 1:32) and determine if they are protected against a lethal orthopoxvirus infection, such as following monkeypox challenge. This experiment would prove or disprove the hypothesis that a serological correlate of protective immunity exists and may lay the foundation for future vaccine design. Of note, the protective level of yellow fever immunity (log 10 ≥ 0.7 neutralizing titer) was also established in non-human primates by simply vaccinating groups of animals with different doses of the yellow fever vaccine, quantitating vaccine-induced antibody levels, and then challenging them with a lethal dose of yellow fever virus [ 49 ]. This serological correlate of protection (which ignores the role of vaccine-induced T cell responses) has been established as the benchmark of protective yellow fever virus-specific immunity for over 30 years and demonstrates the potential for using animal models to correlate protective immunity in humans. Conclusion Traditional smallpox vaccination has lead to the global eradication of smallpox but continues to be used today in an effort to thwart the potential use of smallpox as a biological weapon. Since this vaccine employs the use of a live virus, there is an inherent risk of adverse events, although these are generally quite rare. New generation smallpox vaccine candidates include MVA and other non-replicating poxviruses and although they demonstrate a high degree of safety, their immunogenicity appears to be substantially lower than traditional smallpox vaccination with live vaccinia virus. The role of MVA and traditional smallpox vaccination (or a combination thereof) in future vaccination campaigns has yet to be determined. However, developing a consensus on the definition of what is required for protective immunity and defining an immunological correlate of immunity would aid in the evaluation of current and future vaccine approaches. List of Abbreviations MVA modified vaccinia virus Ankura GMP good manufacturing practices HIV human immunodeficiency virus CEF chicken embryo fibroblasts HPV human papilloma virus FDA Federal Drug Administration JEV Japanese Encephalitis Virus IMV Intracellular mature virus EEV Extracellular enveloped virus ELISA Enzyme-linked immunosorbent assay IFNγ Interferon-gamma ELISPOT Enzyme-linked immunosorbent Spot assay ICCS Intracellular cytokine staining MHC Major histocompatibility complex VIG Vaccinia immune globulin WHO World Health Organization NIBSC National Institute of Biological Standards and Control EU Elisa Units IU International Units Competing Interests OHSU and Dr. Slifka have a financial interest in Najít Technologies, Inc., a company that may have a commercial interest in the results of this research and technology. This potential conflict was disclosed to the OHSU Conflict of Interest in Research Committee and an approved management plan was implemented. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554756.xml |
532389 | Modern Humans Did Not Admix with Neanderthals during Their Range Expansion into Europe | The process by which the Neanderthals were replaced by modern humans between 42,000 and 30,000 before present is still intriguing. Although no Neanderthal mitochondrial DNA (mtDNA) lineage is found to date among several thousands of Europeans and in seven early modern Europeans, interbreeding rates as high as 25% could not be excluded between the two subspecies. In this study, we introduce a realistic model of the range expansion of early modern humans into Europe, and of their competition and potential admixture with local Neanderthals. Under this scenario, which explicitly models the dynamics of Neanderthals' replacement, we estimate that maximum interbreeding rates between the two populations should have been smaller than 0.1%. We indeed show that the absence of Neanderthal mtDNA sequences in Europe is compatible with at most 120 admixture events between the two populations despite a likely cohabitation time of more than 12,000 y. This extremely low number strongly suggests an almost complete sterility between Neanderthal females and modern human males, implying that the two populations were probably distinct biological species. | Introduction The “Neanderthals” or Homo sapiens neanderthalensis (HN) constitute a group of hominids, whose particular morphology developed in Europe during the last 350,000 y under the effect of selection and genetic drift, reaching its final form approximately 130,000 y ago ( Klein 2003 ). This subgroup of hominids populated Europe and western Asia until the arrival of the first modern humans, Homo sapiens sapiens (HS), approximately 45,000 y ago ( Mellars 1992 ). This arrival coincided with the beginning of Neanderthal decline, a process that occurred in less than 15,000 y and that is still not fully understood ( Stringer and Davies 2001 ). An important question which remains to be assessed is whether Neanderthals could hybridize with modern humans and if they left some traces in the current modern human gene pool. While this hypothesis is excluded under the Recent African Origin Model (RAO), which postulates a complete replacement of former members of the genus by H. sapiens, it is central to the tenets of the multiregional hypothesis ( Eckhardt et al. 1993 ; Wolpoff et al. 2000 ), which assumes a gradual transition from H. erectus to modern humans on different continents. From a paleontological and archaeological point of view the debate is still open, even if the supporters of the RAO ( Stringer and Davies 2001 ; Rak et al. 2002 ; Schmitz et al. 2002 ) are gaining momentum over those supporting European regional continuity ( Duarte et al. 1999 ; but see also Tattersall and Schwartz 1999 ). Recent morphological studies support a clear distinction between Neanderthals and modern humans ( Harvati 2003 ; Ramirez Rozzi and Bermudez De Castro 2004 ), and genetic evidence, such as the clear divergence and monophyly of the HN mitochondrial DNA (mtDNA) control region ( Krings et al. 1997 , 1999 ; Ovchinnikov et al. 2000 ), suggested a long separation of the HN and HS female lineages ( Krings et al. 2000 ; Scholz et al. 2000 ; Schmitz et al. 2002 ; Caramelli et al. 2003 ), with a divergence time estimated to lie between 300,000 and 750,000 y ago ( Krings et al. 1997 , 1999 ). The complete absence of Neanderthal mtDNA sequences in the current European gene pool, attested from the study of more than 4,000 recorded sequences ( Richards et al. 1996 ; Handt et al. 1998 ) supported the absence of Neanderthal mtDNA leakage in the modern gene pool, but it was argued that even if some HN genes could have passed in the ancient Cro-Magnon gene pool, they could have been lost through genetic drift ( Relethford 2001 ; Hagelberg 2003 ). Recently, several attempts were made at circumventing the drift problem by the direct sequencing of modern human fossils contemporary with the last Neanderthals. Cro-Magnon sequences were found very similar to those of current Europeans ( Caramelli et al. 2003 ), even though contamination from modern DNA could not be completely excluded ( Serre et al. 2004 ). All studies nevertheless agreed in showing the absence of Neanderthal sequence motifs among early modern human fossil DNA ( Caramelli et al. 2003 ; Serre et al. 2004 ), but only Neanderthal contributions larger than 25% to the modern gene pool could be statistically excluded under a simple model ( Figure 1 A and 1 B) of instantaneous mixing of Neanderthals and modern humans ( Nordborg 1998 ; Serre et al. 2004 ). Thus, the problem of the genetic relationships between Neanderthals and modern humans remains fully open. Figure 1 Different Models of the Interactions between Neanderthals and Modern Humans (A) Model of instantaneous mixing of unsubdivided Neanderthal and modern human populations. (B) Same as (A), but with an exponential growth of the modern human population having started before the admixture with Neanderthals. (C) Model of a progressive range expansion of modern humans into Europe. This model is spatially explicit, and the modern human population occupies a different range than the Neanderthal population before the admixture. Under this model, admixture is progressive and occurs because modern humans move into the territory of Neanderthals, a territory that shrinks with the advance of modern humans. In order to further investigate this issue, we have developed a more realistic modeling of the admixture process between Neanderthals and early modern humans. In brief, the differences with previous approaches are the following (see Figure 1 and the Materials and Methods section for further details): (1) Europe is assumed to be subdivided into small territories potentially harboring two subpopulations (demes): an HN and an HS deme; (2) Europe is settled progressively by modern humans, resulting in a range expansion from the Near East. This range expansion implies also a demographic expansion of early modern Europeans, which stops when Europe is fully settled; (3) local population size is logistically regulated for both Neanderthals and modern humans; (4) we assume there is competition between modern humans and Neanderthals, resulting in the progressive replacement of Neanderthals by modern humans due to their higher carrying capacity caused by a better exploitation of local resources ( Klein 2003 ); (5) Consequently, admixture between the two populations is also progressive and occurs in subdivisions occupied by both populations, in a narrow strip at the front of the spatially expanding modern human population ( Figure 2 ); (6) coalescent simulations are used to estimate the likelihood of different rates of local admixture between modern humans and Neanderthals, given that Neanderthal mtDNA sequences are not observed in current Europeans. Figure 2 Range Expansion of Modern Humans into Europe from the Near East Simulations begin 1,600 generations ago, with the area of Europe already colonized by Neanderthals shown in light gray, and an origin of modern human expansion indicated by a black arrow (lane A). Lanes (B–F) show the progression of the wave of advance of modern humans (dark gray) into Europe at different times before present. The black band at the front of the expansion wave represents the restricted zone of cohabitation between modern humans and Neanderthals. The additional realism of this model makes it also more complex, and the range expansion and admixture processes will depend on several parameters, like the carrying capacities of the local populations, their intrinsic growth rate, the amount of gene flow between adjacent demes, the local rate of admixture between populations, or the geographical origin of the range expansion. Since it is difficult to explore this complex parameter space, we used archeological and paleodemographic information to calibrate the values of these parameters. For instance, the estimated duration of the replacement process (about 12,500 y, Bocquet-Appel and Demars 2000a ) was used to adjust the speed of the expansion of modern humans and, thus, provided strong constraints on local growth and emigration rates. Based on available information, we thus defined a set of plausible parameter values considered as a basic scenario (scenario A). Local admixture rate, which is the parameter of interest here, was then varied, and its effect on the estimated contribution of Neanderthals to the current modern human gene pool was recorded. The sensitivity of admixture estimates to alternative parameterization of our model was studied in eight alternative scenarios (scenarios B to I), by varying each time the values of a few parameters. Results Expected Neanderthal Contribution to the Current European Gene Pool as a Function of Admixture Rates The description of the nine envisioned scenarios for the colonization of Europe by modern humans is reported in Table 1 . For each of these scenarios, the admixture rate, which is the parameter of interest in this study, was allowed to vary and only marginally influenced the cohabitation period and the replacement time of HN by HS ( Table 1 ). Note that the cohabitation period at any given place (shown as a narrow black band on Figure 2 ) is limited to 6–37 generations, depending on the scenario. Table 1 Expected Proportion of Neanderthal Lineages in the Present Modern Human Gene Pool under Different Demographic Scenarios The expected contribution of Neanderthal lineages in the current gene pool of modern humans (over all the simulated demes) was obtained from 10,000 simulations. Standard deviations are shown in italic. Demographic scenarios: (A) The basic scenario with realistic parameters; (B ) identical to (A), with an origin in Iran at the extreme east of the simulated area; (C) identical to (A), but with a diffused source area consisting of 25 demes at K HS = 40, instead of only one deme; (D) identical to (A), but HS occupied all the south of the Neanderthal range (North Africa and North of the Arabian Peninsula) before the onset of the expansion, which corresponds to an HS initial population size of 14,000 breeding females; (E) identical to (A), with r HS = 0.8, and K HN = 25; (F) identical to (A), with m HS = 0.5 and K HN = 25 as in (D); (G) identical to (A) with a faster colonization time, due to larger growth and migration rates ( m HS = 0.35 and r HS = 0.6); (H) identical to (A), with interbreeding resulting in symmetrical transfer of genes between modern humans and Neanderthals; (I) identical to (A), but with carrying capacity K HS being reached instantaneously and a local recruitment of γK HS Neanderthal lineages. In this latter scenario, there is thus a single event of admixture at demographic equilibrium and no logistic growth a K HN : carrying capacity of Neanderthal demes; K HS : carrying capacity of modern human demes; r HS : intrinsic rate of growth of modern humans per generation; m HS : migration rate between adjacent modern human demes b In generation c The different rates of admixture are given in number of admixture events per deme. For instance, a value of 1/10 implies an average of one admixture event for ten demes for the whole period of cohabitation between Neanderthals and modern humans The expected proportion of Neanderthal genes in the gene pool of modern humans was estimated by coalescent simulations and is reported in Table 1 for different rates of admixture between Neanderthals and modern humans. At odds with previous estimates ( Nordborg 1998 ; Gutierrez et al. 2002 ; Serre et al. 2004 ), our simulations show that even for very few admixture events, the contribution of the Neanderthal lineages in the current gene pool should be very large (see also Figure S1 ). For instance, in scenario A, with a 4-fold advantage in exploitation of local resources by modern humans, a single fertile admixture event in one deme out of ten over the whole period of coexistence between HN and HS should lead to the observation of 38% of HN genes in the present mtDNA HS gene pool (scenario A in Table 1 ). This proportion would be lower but still amount to 15% if the advantage of modern humans was reduced to 1.6 times over Neanderthals with the same admixture rate (scenario F in Table 1 ). With higher but still relatively low levels of admixture, a majority of Neanderthal genes should be expected in the current European gene pool ( Table 1 ). For instance, with as much as two admixture events per cell over the total coexistence period of Neanderthals and modern humans, more than 95% of the current HS gene pool should be tracing back to Neanderthals, for all scenarios with logistic demographic regulation described in Table 1 (scenarios A to H). As shown on Figure 3 , the proportion of current lineages that can be traced to Neanderthals is, however, not uniformly distributed over Europe in scenario of moderate or low interbreeding. A gradient should be visible from the source of the range expansion (which shows the largest proportion of modern human genes) toward the margins of the expansion (the British Isles and the Iberian Peninsula), which should then be expected to harbor a larger proportion of Neanderthal genes than the rest of Europe ( Figure 3 ). However, this gradient would be relatively weak, and the expected proportion of HN lineages at any position is primarily affected by the degree of admixture between the two populations. Figure 3 Expected Proportion of Neanderthal Lineages (in Black) among European Samples under Demographic Scenario A ( Table 1 ) at Different Geographic Locations, for Different Interbreeding Rates (A) One admixture event on average per 50 demes over the whole period of cohabitation between Neanderthals and modern humans; (B) one admixture event per five demes; (C) one admixture event per two demes; (D) one admixture event per deme. The finding that even minute amounts of interbreeding between Neanderthals and modern humans should lead to a massive introgression of Neanderthals' mtDNAs into the Cro-Magnon gene pool is somehow counterintuitive and deserves further explanations. The successful introgression of Neanderthal mtDNAs is due to a massive dilution of the modern human mtDNA gene pool into that of the pre-existing population ( Chikhi et al. 2002 ) and to a low probability of being lost by drift at the time of introgression (see below). The dilution process can be seen as follows: An HN gene entering the HS gene pool at an early stage of the colonization process will lower the frequency of HS genes in the HS deme; the migrants sent from this deme to colonize an adjacent new territory can themselves harbor HN genes, so that a further HS deme can be founded by a mixture of HS and HN genes; additional admixture events will further lower the proportion of HS genes in HS demes. The repetition of these admixture and migration steps will thus rapidly dilute HS genes. Under this process, the European HS population can be fully introgressed by HN genes under scenarios A to H, if two or more admixture events occurred in each deme (see Table 1 , last two columns). For such large rates of admixture, the fraction of HS genes in demes adjacent to the source of HS expansion is already diluted by more than 28% with HN genes (results not shown). Therefore, in the absence of counteracting selective forces, the dilution process repeated over several demes would hinder the spread of HS genes away from the source of the colonization. The range expansion would thus be mainly carried out by individuals having HN genes, explaining why the HS European population would appear fully introgressed by HN genes. The success of introgressing HN genes is also due to their integration into the HS deme while it is in a period of demographic (logistic) growth (see Figure S2 ), so that these introgressing genes are unlikely to be lost by genetic drift, and will, rather, be amplified by the logistic growth process occurring in the HS deme. In order to assess the importance of the period of logistic growth relative to the dilution process, we have modeled a range expansion process where a newly founded deme reaches instantaneously its carrying capacity, and where a given proportion of genes is recruited from the local Neanderthal gene pool. The results of those simulations (reported in Table 1 as scenario I) show that without logistic growth much larger interbreeding rates would be necessary to have the same impact on current human diversity. Indeed, the occurrence of two admixture events per deme over the whole cohabitation period would only lead to 5% of the current gene pool being of Neanderthal ancestry, instead of 100% when logistic growth is implemented. Estimation of Admixture Rates between Neanderthals and Modern Humans The present results show that if Neanderthals could freely breed with modern humans, having progressively invaded their territory, their contribution to our gene pool would be immense. Since no Neanderthal mtDNA sequence has been observed so far among present Europeans, it is of interest to estimate the maximum admixture rate between Neanderthals and modern humans that would be compatible with an absence of Neanderthal genes, accounting for the current sampling effort and genetic drift over the last 30,000 y. This assessment was done by coalescent simulations. The likelihoods of different admixture rates are reported in Figure 4 for each scenario. Maximum-likelihood estimates are obviously obtained for a total absence of interbreeding between HS and HN, but here the interest lies in the upper limit of a 95% confidence interval. We see that the scenarios A to H can be divided into three groups. Scenarios A, C, G, and H lead to very similar upper bounds for the estimation of the maximum admixture rate (approximately 0.015 admixture events per deme; see Table 2 ). Similarity of results obtained for scenarios A and C show that the fact that the origin of the spread of modern humans was diffused over a large area or concentrated at a single point does not substantially influence our results. A shorter duration of the colonization of Europe by HS (approximately 8,000 y; scenario G) leads to an estimation very similar to that obtained under scenario A. Also the implementation of fully symmetric interbreeding between HN and HS (scenario H) leads to results almost identical to those obtained when we only allow breeding between HN females and HS males (scenario A). The place of origin for modern humans seems more important, as a putative origin in Iran (scenario B) or in North Africa (scenario D) leads to even lower maximum interbreeding rates (approximately 0.01 admixture events per deme) than if the source is located closer to Europe as in scenario A. Moreover, scenario D also shows that a much larger initial size of the HS population (14,000 breeding females instead of 40 in scenario A) does not reduce the final Neanderthal contribution to the HS gene pool. This is because we model local (at the deme level) and not global contacts between the two populations. Finally, scenarios E and F, corresponding to larger carrying capacities of Neanderthals, would be compatible with a larger amount of admixture between the two species (approximately 0.03 admixture events per deme), which is understandable given the longer cohabitation times under these scenarios (21–37 generations) than under scenarios A–D and G–H (6–12 generations). The estimates of the average number of admixture events per deme can be translated into a maximum number of interbreeding events having occurred over all Europe during the whole replacement process of Neanderthals by modern humans, as reported in Table 2 . We find that, depending on the scenario, these maximum estimates range between 34 (scenario B) and 120 (scenario E) admixture events over the whole of Europe , which are extremely low values given the fact that the two populations have certainly coexisted for more than 12,000 y in that region. Figure 4 Likelihood of Different Rates of Interbreeding under the Nine Scenarios Described in Table 1 The horizontal bold dashed line corresponds to 14.7% of the maximum likelihood, defining the upper limit of a 95% confidence interval for the interbreeding rates (see, e.g., Kalbfleisch 1985 ). Table 2 Measure of Genetic Interaction between Neanderthals and Modern Humans a Upper limit of a 95% confidence interval b This figure is computed from the previous column by assuming that there were a total of 140,000 reproducing females in the total modern human population in Europe ( see Materials and Methods ) Discussion Our simulations show that the mitochondrial evidence in favor of no, or very little, interbreeding between Neanderthals and modern humans is much stronger than previously realized ( Wall 2000 ; Nordborg 2001 ). We indeed find that the current absence of Neanderthal mtDNA genes is compatible with a maximum admixture rate about 400 times smaller than that previously estimated ( Nordborg 1998 ; Serre et al. 2004 ). This initial estimate (25%) was, however, based on a simple but unrealistic model of evolution, assuming no population subdivision, constant population size, and a single and instantaneous admixture event between Neanderthals and modern humans. Taking into account the progressive nature of the range expansion of modern humans into Europe, the maximum initial input of Neanderthal genes into the Paleolithic European population can thus be estimated to lie between only 0.02% (scenario B) and 0.09% (scenario E) ( Table 2 ). Our simulations of alternative scenarios of HS range expansion into Europe suggest that our results are not very sensitive to local HS growth rates, level of gene flow between neighboring HS demes, or the geographical origin of HS range expansion. It is also worth emphasizing that the final HN contribution to the European gene pool does not really depend on the size and spread of the population at the source of the range expansion (compare scenario A to C and D in Table 1 ). This is logical since the colonization process starts from a restricted number of demes at the edge of the pre-existing range in our model of subdivided population (see Figure 1 C). If this model is correct, it implies that the current European genes should have coalesced in a small number of individuals present in the demes at the source of the colonization of Europe, or, in other words, that there was a bottleneck having preceded the range expansion into Europe. Available data on European mtDNA diversity indeed support this view, since most European populations do present a signal of Paleolithic demographic expansion from a small population, which could be dated to about 40,000 y ago ( Excoffier and Schneider 1999 ). Additional complexities of the simulation model could have been envisioned, like the possibility for long-range dispersal, some heterogeneity of the environment leading to different carrying capacities and preferential colonization routes, or uneven migration rates. However, these extra parameters would have been very difficult to calibrate due to the scarcity of paleodemographic data. Moreover, it is likely that they would not have lead to qualitatively different results. For instance, since long-range dispersal speeds up the colonization process ( Nichols and Hewitt 1994 ), short range migration rates would need to be reduced, in order to preserve a realistic colonization time. But this reduction would have no effect on local cohabitation time, which is the important factor affecting admixture rates ( Table 2 ). Another source of realism could be the implementation of a recent Neolithic expansion wave on top of a Paleolithic substrate. This additional expansion wave has not been implemented here, as it is clearly beyond the scope of the present study. However, our present results suggest that small amounts of admixture between the Paleolithic and the Neolithic populations would lead to a massive contribution of Paleolithic lineages among the current Europeans. This point is important as it implies that if Neanderthal lineages had been present among the Paleolithic populations, they would not have been erased by the spread of the Neolithic in Europe. If we were using previous estimations of the Neolithic contribution to the current European genetic pool of about 50% ( Barbujani and Dupanloup 2002 ; Chikhi 2002 ), the effect of a Neolithic expansion would require our estimates of the initial input of HN into the modern pool to be roughly multiplied by two, but still be very small (0.07% for scenario A). Note also that the simulation of a pure acculturation process, which amounts to increasing the carrying capacity of populations after the Neolithic by a factor 250 has virtually no effect on the expected proportion of Neanderthal genes in current Europeans (see Figure S1 ). Another argument against a major influence of the Neolithic expansion stems from mtDNA studies, since the demographic expansion inferred from mtDNA diversity and dated to about 40,000 y ago ( Excoffier and Schneider 1999 ) implies that most of the mtDNA lineages of current Europeans result from a Paleolithic range expansion ( Ray et al. 2003 ). If the expansion of Neolithic settlers had fully erased Paleolithic mtDNA diversity, one would indeed not expect to see this Paleolithic expansion signal. It thus argues in favor of a minor contribution of Neolithic genes to the current European gene pool, as expected under our model of progressive range expansion with continuous mixing. Compared to previous models assuming an instantaneous mixing of HN and HS populations ( Nordborg 1998 ; Serre et al. 2004 ) (see Figure 1 A), we find that extremely small Neanderthal contributions should still be visible in the European gene pool. It implies that HN genes have a much larger probability of persisting when entering a progressively invading HS population than when entering a stationary population. This is because HN genes enter the HS population in demes that are still growing in size (see Figure S2 ), which prevents them from being lost by genetic drift and which amplifies their absolute number in the deme, making it likely they will persist and reach observable frequencies in the global population. This process is actually similar to that occurring in an unsubdivided growing population (e.g., Otto and Whitlock 1997 ). Actually, if HN genes were to directly enter an unsubdivided HS population that grew exponentially until today (see Figure 1 B), the current absence of HN genes would also imply a very small amount of Neanderthal introgression into our gene pool ( Nordborg 1998 ; Serre et al. 2004 ). However, this continuous and global exponential growth process appears difficult to justify ( Serre et al. 2004 ) and does not really apply to the late Pleistocene human population ( Weiss 1984 ; Biraben 2003 ). Under our model, the progressive range expansion ( Figure 1 C) and the local logistic growth contribute to reduce the probability of losing introgressed HN genes. Without logistic growth, much larger interbreeding rates would be necessary to have the same impact on current human diversity (see scenario I in Table 1 and in Figure 4 ). Under this scenario, the absence of Neanderthal mtDNA sequences in present Europeans is still compatible with a maximum of about 1,850 fertile breedings between Neanderthal females and Cro-Magnon males, corresponding to a maximum initial input of 1.2% Neanderthal genes into the European Cro-Magnon population ( Table 2 ). This figure being 20 times larger than when assuming an initial logistic growth of newly founded populations, it shows that the local logistic growth and the progressive range expansion contribute equally to reducing the inferred admixture rate compared to the simple model assuming a single admixture event and an instantaneous settlement of Europe by modern humans (see Figure 1 A) ( Serre et al. 2004 ). However, because new territories are often colonized by a few migrants and not by whole populations, local logistic growth has been incorporated into most models of range expansion (e.g., Fisher 1937 ; Skellam 1951 ; Shigesada and Kawasaki 1997 ). It should thus be considered as a normal feature of range expansions. Another important result of this study is to show that an expanding population or species is likely to have its own genome invaded by that of the invaded population if interbreeding is possible and gradual, which could explain some documented cases of mtDNA introgression (e.g., Bernatchez et al. 1995 ; Shaw 2002 ). Our results indeed suggest that introgression should occur preferentially in species having gone through a range expansion, and that the introgressing genome would be that of the invaded population and not that of the invasive species. Of course this result should only apply to the part of the genome that is not under selection or that is not linked to the selective advantage of the invaders. If the mitochondrial genome of modern humans was involved in their higher fitness, the absence of observed mtDNA introgression would not necessarily be due to an absence of interbreeding, but would rather result from an active selection process against crosses between Neanderthal females and modern human males, and one would therefore expect to see potential leakage of Neanderthal genes in our nuclear genome. While some evidence for the differential fitness of some mtDNA human genomes in distinct climates has been recently found ( Mishmar et al. 2003 ; Ruiz-Pesini et al. 2004 ), it is unlikely that such differences were involved in the selective advantage of modern humans over Neanderthals. It is indeed doubtful that modern humans coming from the Middle East would have had mitochondria better adapted to the colder environment of Europe than Neanderthals, who had spent tens of thousands of years in such a climate ( Tattersall and Schwartz 1999 ; Klein 2003 ). It is therefore more likely that modern humans' higher technology and higher cognitive abilities ( Klein 2003 ), resulting in better resource processing and environmental exploitation, have allowed them to out-compete Neanderthals, and that mtDNA was selectively neutral in that respect. It should however be kept in mind that our conclusions assume no sex bias in interbreeding rates. Studies of fossil Y chromosome or nuclear DNA would be needed to examine the basis of this assumption, but it seems difficult to imagine why interbreeding between Neanderthal men and modern human females resulting in the incorporation of Neanderthal genes would have been more frequent than the reverse situation. Even though our model of interaction and competition between Neanderthals and modern humans may not entirely correspond to the reality, it captures two important historical aspects that were neglected in previous studies. The first one is the documented progressive spread of modern humans in Europe (see Figures 1 and 2 ), and the second is the local and progressive demographic growth of Paleolithic populations, with density-dependent interactions with Neanderthals. The incorporation of these additional sources of realism cannot be handled by current analytical models, but it can be readily integrated into a coalescent simulation framework, showing that it will be possible in the future to predict patterns of molecular diversity among populations or species belonging to a particular ecological network. Given the long period of cohabitation of the two populations in Europe and ample opportunities to interbreed, the absence or extremely low number of admixture events between Neanderthals and modern humans is best explained by intersterility or reduced fitness of hybrid individuals, promoting these populations to the status of different biological species. No interbreeding between the two populations also strongly argues in favor of a complete replacement of previous members of the genus Homo by modern humans and against a multiregional evolution of H. sapiens ( Eckhardt et al. 1993 ; Wolpoff et al. 2000 ). It thus gives more credit to the RAO hypothesis ( Excoffier 2002 ; Stringer 2002 ), since some very divergent H. erectus mitochondrial sequences should have also been observed if interbreeding had occurred during the colonization of Eurasia by modern humans from Africa. Our conclusions about the genetic incompatibility between modern humans and Neanderthals would however be wrong if the absence of Neanderthal mtDNA genes in the current gene pool of modern Europeans was due to some processes that were not incorporated into our model. For instance, a range expansion of Neolithic populations without genetic contacts with Paleolithic could have erased both Paleolithic and remaining Neanderthal genes, but as discussed above, there are evidences for a substantial contribution of Paleolithic populations to the current gene pool ( Barbujani and Dupanloup 2002 ; Chikhi et al. 2002 ; Dupanloup et al. 2004 ), invalidating this theory. Also an extremely rapid range expansion of a very large and unsubdivided modern population would also be compatible with an absence of Neanderthal genes despite considerable admixture, like in the scenario shown in Figure 1 A ( Nordborg 1998 ; Serre et al. 2004 ), but the long duration of the replacement process would be difficult to justify in that case. Finally, the occurrence of a cultural or ecological barrier, and not necessarily of a genetic barrier, could have prevented the realization of biologically possible hybridizations. Under this scenario, Neanderthals and early modern humans would have just avoided each other, which is contradicted by the observation of technological exchanges between Neanderthals and Cro-Magnons (e.g., Hublin et al. 1996 ). Moreover, the fact that the two populations had a very similar economy ( Klein 1999 , p. 530), indicates they had occupied an overlapping ecological niche and had thus ample opportunities to meet. It therefore seems that our model of subdivided population and progressive range expansion, implying local contacts, competition, and potential hybridization is quite plausible. One of its merits is also to explain both the replacement of Neanderthals by modern humans through a better exploitation of local resources, but also the late colonization of Europe by modern humans, which would have been possible only after the emergence of refined Upper Paleolithic technologies giving a competitive edge over Neanderthal industries ( Klein 1999 , pp. 511–524). Materials and Methods Digital map of Europe The simulated region corresponds to the geographical region encompassing Europe, the Near East and North Africa. It has been modeled as a collection of 7,500 square cells of 2,500 km 2 each, arranged on a two-dimensional grid, with contours delimited by seas and oceans. Each cell harbors two demes, one potentially occupied by modern humans (HS) and one potentially occupied by Neanderthals (HN). Given the estimated range distribution of Neanderthals ( Klein 2003 ), HN demes were allowed in only 3,500 cells, mainly located in the lower part of Europe and in the Near East (see Figure 2 A). Three land bridges have been artificially added to allow the settlement of Great Britain and Sicily. Simulation of the colonization of Europe by modern humans The simulation of the colonization process in Europe is an extension of that described in absence of competition in a homogeneous square world ( Ray et al. 2003 ). At the beginning of the simulation, 1,600 generations ago (corresponding to 40,000 y ago when assuming a generation time of 25 y), the HN demes are all filled at their carrying capacity, K HN , and, in the basic scenario, the population HS is assumed to be restricted to a single deme in the Near East at a position corresponding approximately to the present border between Saudi Arabia and Jordan. Note that alternative locations and a more widespread distribution are also envisioned in other scenarios (see Table 1 ).This source for the spatial and demographic expansion of modern humans into Europe has been chosen arbitrarily, as its exact origin is still debated ( Bocquet-Appel and Demars 2000a ; Kozlowski and Otte 2000 ). Since we model the evolution of mtDNA, we only simulate the spread of females, but we implicitly assume that there are the same number of males and females in each deme. The source deme for HS is assumed to be at its carrying capacity K HS of 40 females, corresponding to a density of about 0.06–0.1 individuals per km 2 (including males and juveniles), in agreement with density estimates for Pleistocene hunter-gatherers ( Steele et al. 1998 ; Bocquet-Appel and Demars 2000b ). HS individuals can then migrate freely to each of the four neighboring HS demes at rate m /4. When one or more HS individuals enter an empty deme, it results in a colonization event, which initiates a local logistic growth process, with intrinsic rate of growth r HS per generation, and with limiting carrying capacity K HS . Interactions between the HS and the HN demes of the same cell are described below in more detail, and its combination with migrations between HS demes results in a wave of advance progressing from the Near East toward Europe and North Africa. Demographic model incorporating competition and admixture We describe here a demographic model of interaction between populations, incorporating competition and interbreeding between individuals of the HN and HS populations, as well as migration between neighboring demes from the same subdivided population. We distinguish here migrations events between HN and HS populations from migrations between neighboring HN or HS populations. We model the former ones as admixture events, whereas the latter ones correspond to true dispersal events. The life cycle of a population at a given generation is as follows: admixture, logistic regulation incorporating competition, followed by migration. This life cycle thus assumes that migration is at the adult stage. In line with previous work ( Barbujani et al. 1995 ), the frequency of admixture events is assumed to be density-dependent. Within a given deme, each of the N i individuals from the i-th population has a probability to reproduce successfully with one of the N j members of the j-th population, and γ ij represents the probability that such a mating results in a fertile offspring. Alternatively, γ ij could represent the relative fitness of hybrid individuals or an index of disassortative mating. Following admixture, population densities are then first updated as Our model of density regulation incorporating competition is based on the Lotka–Volterra interspecific competition model, which is an extension of the logistic growth model ( Volterra 1926 ; Lotka 1932 ). For each population, a new density N ″ i is calculated from the former density as where r i is the intrinsic growth rate of the i -th population, K i is its carrying capacity, and α ij is an asymmetric competition coefficient ( Begon et al. 1996 , pp. 274–278). An α ij value of 1 implies that individuals of the j -th population have as much influence on those of population i as on their own conspecific, or that competition between populations is as strong as competition within a population. Lower values of α ij indicate lower levels of competition between populations than within populations; a value of zero implies no competition between individuals from different populations. We have decided here not to fix α ij values, but to make them density-dependent as reflecting the fact that the influence of the members of a population on the other grows with its density. An example of the demographic transition between HN and HS is shown in Figure S2 , together with the amount of admixture between the two populations. In the migration phase, each population of each deme can send emigrants to the same population in neighboring demes at rate m. N ″ i m emigrants are thus sent outward each generation, and distributed equally among the four neighboring demes, as described previously ( Ray et al. 2003 ). If a gene is sent to an occupied deme, the migration event results in gene flow; otherwise, it results in the colonization of a new deme. This latter possibility only exists for the population of modern humans, since we assume that Europe was already fully colonized by Neanderthals. Finally, the densities of the two populations are updated as a balance between logistic growth, migration, and admixture as where I i is the number of immigrants received from neighboring demes. Parameter calibration We have calibrated the parameters of our simulation model from available paleodemographic information and from the estimated colonization time of Europe by modern humans. Estimates of the total number of hunter-gatherers living before Neolithic times range between 5 and 10 million ( Coale 1974 ; Hassan 1981 ; Weiss 1984 ; Landers 1992 ; Chikhi et al. 2002 ), of whom about 1 million individuals were living in Europe. Taking a carrying capacity K HS of 40 females would imply the presence of 220,000 effective mtDNA genes in the 5,500 demes occupied by modern humans in Europe and the Middle East. Since this number represents only females, the total number of individuals living over Europe was multiplied by four to include men and juveniles, leading to a total density of about 880,000 HS individuals. This value of K HS corresponds to a density of 0.064 individuals per square kilometer, which is close to the value (0.04) used by some previous simulation of modern humans ( Rendine et al. 1986 ; Barbujani et al. 1995 ) and well within the range obtained from actual hunter-gatherer groups (0.01–0.35; Binford 2001 ) or that estimated for ancient hunter-gatherers (0.015–0.2; Steele et al. 1998 ; Bocquet-Appel and Demars 2000b ). The time required for the colonization of Europe by modern humans is the other information that was used to calibrate the growth rates, r HS , the rate of migration, m HS , and the Neanderthal carrying capacity (K HN ), as these three parameters have an influence on the speed of the migration wave ( Fisher 1937 ; Skellam 1951 ). Since modern humans arrived in Europe approximately 40,000 y ago and occupied the whole continent by 27,500 before present (BP) ( Bocquet-Appel and Demars 2000b ), the colonization process lasted approximately 500 generations, assuming an average generation time of 25 to 30 y ( Tremblay and Vezina 2000 ; Helgason et al. 2003 ). Scenarios of modern human range expansion in Europe Among the many sets of parameter values leading to the appropriate colonization time and the complete disappearance of Neanderthals, we have retained the following scenarios. Scenario A: Origin of HS in a single deme of the Near East at the border between Saudi Arabia and Jordan, m HS = m HN = 0.25, r HN = 0.4, and K HN = 10, r HS = 0.4, K HS = 40. Note that a value of K HN of ten corresponds to a total density of about 140,000 Neanderthals over Europe (0.016 individuals per km 2 ), which is of the same order of magnitude as the rare available estimates (250,000 Neanderthals, Biraben 2003 ). Under this scenario, we have only considered admixture events between HN females and HS males, such that γ HS,HN = 0 . Eight alternative scenarios have been considered by using extreme values of the parameters of the model ( m, r, K, European colonization time, place, and size of initial HS population). Scenario B is identical to scenario A, except that the HS origin is located in Iran. Scenario C uses the same parameters as scenario A, but the HS source is more diffuse and corresponds to a subdivided population of 25 demes (1,000 breeding females) surrounding the source deme defined in scenario A. Scenario D is identical to A, except that the initial HS population is even much more numerous (14,000 breeding females located in 1,400 demes) and occupies all the south area of the HN occupation zone. Scenario E is identical to A, but r HS is here equal to 0.8, which is the maximum growth rate estimated for the Paleolithic human population ( Ammerman and Cavalli-Sforza 1984 ; Young and Bettinger 1995 ). Scenario F is identical to A, except that m HS is here much higher and equal to 0.5, implying that 50% of the women are recruited in adjacent demes. The carrying capacity of Neanderthals K HN had to be readjusted for scenarios E and F, which may appear as extreme, in order to maintain a colonization time of about 500 generations. It was indeed set to 25, giving a total density of HN of 350,000 individuals over Europe. Scenario G is identical to A, except that r HS is here equal to 0.6 and m HS is equal to 0.35, leading to a shorter colonization time of the European continent by HS. Under scenario G, the colonization time of Europe is approximately 8,000 y, which would correspond to the minimum colonization time estimated from direct fossil evidence, since the first European HS fossil is dated to about 36,000 y BP ( Trinkaus et al. 2003 ), and the latest HN is dated around 28,000 y BP ( Smith et al. 1999 ). Scenario H is identical to A, but admixture can occur between HN males and HS females as well, such that γ HS,HN = γ HN,HS . Finally, scenario I uses the same parameters as A, but a different demographic model. When a cell is colonized by HS, it is directly filled at K HS with an initial proportion γ of Neanderthals. Admixture thus occurs when demographic equilibrium is already reached, and not during the demographic growth as in the other models. While the γ values are the true parameters of our model, they may not be very telling per se, and we have therefore chosen to quantify levels of interbreeding between populations using another parameterization, which is the average number of admixture events per deme between modern humans and Neanderthals. By performing a large series of simulations, we could find the values of γ leading to a given average number of admixture events per deme (e.g., 1/500, 1/100, 1/10, 1, 2, etc.). For instance, a value of 1/10 means that one admixture event occurred on average in one deme out of ten during the whole cohabitation period between HN and HS. Coalescent simulations For each scenario and for different interbreeding values, γ ij , the demography of the more than 14,000 demes is thus simulated for 1,600 generations. The density of all demes, the number of migrants exchanged between demes from the same population, and the number of admixture events resulting in gene movements between Neanderthals and modern humans are recorded in a database. This demographic database is then used to simulate the genealogy of samples of 40 genes drawn from 100 demes, representing a total of 4,000 modern human genes distributed over all Europe and corresponding approximately to the current sampling effort of European mtDNA sequence ( Richards et al. 1996 ; Handt et al. 1998 ). The coalescent simulations proceed as described previously ( Ray et al. 2003 ; Currat et al. 2004 ). The average proportion of sampled genes whose ancestors can be traced to some Neanderthal lineages was then computed over 10,000 simulations. The likelihood of each interbreeding coefficient, γ ij , is estimated for the different scenarios by the proportion of 10,000 simulations that lead to a Most Recent Common Ancestor of all 4,000 sampled mtDNA sequences being of modern human origin. Supporting Information Figure S1 Proportion of Neanderthal Lineages in the European Population as a Function of the Average Number of Admixture Events per Deme between HN and HS These values are given for the nine scenarios (A–I) listed in Table 1 , and for a new scenario A+Neol. This latter scenario is similar to A, except that the carrying capacity of the modern humans is increased by a factor 250 at the time of the Neolithic transition (320 generations BP). The influence of this demographic increase on the simulated HN proportion is very weak, as shown on this figure. (357 KB TIF). Click here for additional data file. Figure S2 Evolution of the densities of demes HN (in black) and HS (in gray) within a cell simulated under demographic scenario A for γ ij = 0.4. The cell is colonized by HS at time −1520 ( 0 = present). The thin black line with white circles represents the distribution of admixture events, whose numbers are reported on the right axis (322 KB TIF). Click here for additional data file. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC532389.xml |
522818 | Cytokine and immunoglobulin production by PWM-stimulated peripheral and tumor-infiltrating lymphocytes of undifferentiated nasopharyngeal carcinoma (NPC) patients | Background Undifferentiated Nasopharyngeal Carcinoma (NPC) patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV) which is regularly associated with this tumor. However, no EBV-specific cytotoxic activity is detectable by the standard chromium-release assay at both peripheral and intratumoral levels. The mechanisms underlying this discrepancy between the humoral and cellular immune responses in NPC are still unknown, but might be related to an imbalance in immunoregulatory interleukin production. In this report, we investigated the ability of peripheral (PBL) and tumor- infiltrating (TIL) lymphocytes of undifferentiated NPC patients to produce in vitro three interleukins (IL-2, IL-6, IL-10) and three immunoglobulin isotypes (IgM, IgG, IgA). Methods Lymphocytes from 17 patients and 17 controls were cultured in the presence of Pokeweed mitogen (PWM) for 12 days and their culture supernatants were tested for interleukins and immunoglobulins by specific enzyme-linked immunosorbent assays (ELISA). Data were analysed using Student's t-test and probability values below 5% were considered significant. Results The data obtained indicated that TIL of NPC patients produced significantly more IL-2 (p = 0,0002), IL-10 (p = 0,020), IgM (p= 0,0003) and IgG (p < 0,0001) than their PBL. On the other hand, patients PBL produced significantly higher levels of IL-2 (p = 0,022), IL-10 (p = 0,016) and IgM (p = 0,004) than those of controls. No significant differences for IL-6 and IgA were observed. Conclusion Taken together, our data reinforce the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. An increased ability to produce cytokines such as IL-10 may underlie the discrepancy between humoral and cellular immune responses characteristic of NPC. | Background Undifferentiated nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor characterized by a heavy infiltration of non malignant lymphocytes and most of these tumor infiltrating lymphocytes (TIL) have been shown to be T cells [ 1 ]. The Epstein-Barr virus (EBV) is causally associated with this malignancy since viral DNA is regularly present in the malignant epithelial cells but not in the neighbouring normal tissues. In addition, NPC patients show a specific pattern of humoral responses against EBV antigens [ 2 ]. Viral proteins known to be expressed in NPC tumor cells are the EBV-encoded nuclear antigen 1 (EBNA-1) and the latent membrane proteins LMP-1 in 35 to 65% of cases, and LMP-2 [ 3 , 4 ]. The latent membrane proteins have been shown to serve as targets for EBV-specific cytotoxic T lymphocytes (CTL) from normal seropositive individuals [ 5 , 6 ]. Recently, CD8 positive EBV-specific cytotoxic T cell clones were isolated from the peripheral blood and tumors of NPC patients [ 7 ]. The majority of the isolated CTL clones are directed towards the most immunogenic EBNA3 proteins which are not expressed in NPC tumor cells. No EBV-specific CTL activity is detectable by the standard chromium release assay in NPC patients [ 8 - 10 ] and the activity of any CTLs that would be present in such patients appears to be somehow suppressed. This lack of cytotoxic activity is in sharp contrast with the strong anti-EBV humoral immune response seen in patients [ 11 , 12 ]. The discrepancy between these two types of immune responses in NPC is still unexplained. It has been hypothesized that some viral gene products might have the capacity to influence cytokine production in such a way as to inhibit specific CTL activity [ 3 , 13 ]. Interestingly, the product of the EBV BCRF1 open reading frame has been found to display extensive homology with human interleukin 10 [IL-10 ; [ 14 ]]. Like its human counterpart, this viral product designated vIL-10, exerts immunosuppressive functions [ 15 ]. It is postulated that IL-10 production in malignant tumors may facilitate their escape from immune surveillance [ 16 ]. The expression of IL-10 in NPC has been controversial. While it has been reported that IL-10 is not expressed by NPC cells as detected by RNA in situ hybridisation [ 17 ], some reports using immunohistochemical and molecular techniques showed the expression of this cytokine by epithelial NPC tumor cells and TIL [ 18 - 20 ]. These authors suggested IL-10 as a possible evasion mechanism against the host antiviral system. Such a mechanism would explain the lack of detection of EBV specific cytotoxic activity in NPC patients at both peripheral and intratumoral levels [ 8 - 10 , 21 ]. Indeed, IL-10 is known to inhibit cell-mediated immune responses [ 22 ]. IL-10 is also known for upregulating the B cell response [ 23 ] and therefore, this putative mechanism is in accordance with the strong EBV-specific humoral immune response seen in NPC [ 11 , 12 , 24 ]. Other interleukins such as IL-2 and IL-6 may also appear to be involved in this discrepancy between humoral and cellular immune responses due to their central regulatory effects on T or B cells [ 25 , 26 ]. In this report, we investigated the ability of both peripheral blood lymphocytes (PBL) and TIL of undifferentiated NPC patients to express three interleukins (IL-2, IL-6, IL-10) and three immunoglobulin isotypes (IgM, IgG, IgA) following pokeweed mitogen (PWM) stimulation in vitro. The data obtained indicated some significant differences between NPC patients and controls in interleukin and immunoglobulin production. However, further investigations are needed to establish the relevance of these differences to the discrepancy between humoral and cellular immune responses characteristic of NPC. Methods Patients and controls 17 untreated Tunisian NPC patients were included in this study. Informed consent was obtained from all patients before collection of blood and biopsy samples. These patients presented for treatment at the National Cancer Institute Salah Azaiez, Tunis or at the Farhat Hached hospital, Sousse, Tunisia. Their mean age was 47,5 years (range 25–70). All patients were diagnosed histopathologically as undifferentiated NPC. 17 healthy EBV carriers who presented to donate blood at the National Blood Transfusion Centre, Tunis, Tunisia, were included as controls in this study. Sera of all patients and controls were titrated for antibodies directed against EBV antigens by indirect immunofluorescence [ 27 ]. Lymphocyte preparations Peripheral blood and biopsy samples were taken on the same day from each of the 17 NPC patients. Peripheral blood was collected from patients and healthy donors in heparin. Mononuclear cells were isolated by centrifugation over Ficoll-Hypaque (Pharmacia) according to Boyum [ 28 ]. Following three washes in RPMI-1640 medium (Gibco), cells were resuspended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS ; Gibco) and 50 μg/ml Gentamicin (complete medium) at a concentration of 2 × 10 6 cells/ml. For the preparation of tumor-infiltrating lymphocytes, biopsies were collected from NPC tumors under sterile conditions and immediately transferred to RPMI-1640 medium supplemented with 50 μg/ml Gentamicin. The biopsies were washed several times in this medium and then minced into small pieces in order to extract lymphocytes. The extracted cells were then washed by centrifugation in the same medium and then resuspended in complete medium at a final concentration of 2 × 10 6 cells/ml. Samples from each lymphocyte preparation were taken for immunophenotyping by indirect membrane immunofluorescence using mouse monoclonal antibodies directed against CD3, CD4, CD8 or CD19 and FITC-conjugated goat anti-mouse F(ab') 2 fragment. The labelled cells were counted under a fluorescence microscope. Stimulation by Pokeweed mitogen PBL from healthy donors or NPC patients and TIL from the same patients suspended at 2 × 10 6 cells/ml in complete medium were distributed at 1 ml / well in 24-well Costar plates. One hundred microliters of a predetermined optimal dilution of PWM (Gibco), for treated wells, or complete medium alone, for untreated controls, were added in corresponding wells. The cells were then cultured for 12 days at 37°C, 5% CO 2 and 98% relative humidity in a CO 2 incubator. At the end of this incubation period, culture supernatants were harvested and used for cytokine and immunoglobulin determination as described below. Cytokine and Immunoglobulin determination Culture supernatants of PBL and TIL stimulated by PWM under optimal conditions were used for both cytokine and immunoglobulin determination by enzyme-linked immunosorbent assays (ELISA). IL-2, IL-6 and IL-10 concentrations were measured using commercial sandwich-type ELISA kits (Immunotech enzyme immunoassays, France) according to the procedures described by the manufacturer. IgM, IgG and IgA concentrations were measured using sandwich-type ELISA assays prepared in our laboratory. Briefly, 96 well-microplates (Greiner, Germany) were coated for 2 hours at 37°C and overnight at 4°C with 150 μl/well of isotype-specific mouse monoclonal antibody (Sigma, France) appropriately diluted in 0,1 M carbonate buffer, pH 9.6. The microplates were washed four times with phosphate-buffered saline pH 7.4 containing 0,1 % Tween 20 (PBS-Tween), then blocked with 300 μl/well of PBS containing 1% FCS for 30 minutes at 37°C. For the assay, 100 μl /well of appropriately diluted culture supernatants in PBS-Tween containing 10% FCS were incubated in coated microplates for 2 hours at 37°C. The microplates were then washed four times with PBS-Tween and 100 μl/well of the appropriate alkaline phosphatase-labeled conjugate (Goat anti-human IgM, IgG or IgA, Sigma-France) were added at a 1:20000 dilution in PBS containing 1% FCS. After a two-hour incubation at 37°C, the microplates were washed and a p-nitrophenylphosphate substrate solution was added. The microplates were incubated for 1 hour at 37°C and the reaction was stopped with 50 μl/well of 1 N NaOH. Immunoglobulin standards were generated using purified human immunoglobulins of each isotype (Sigma, France). The microplates were read at 405 nm on a microplate reader (LP-400, Diagnostic Pasteur). Data were represented as the mean immunoglobulin concentration of triplicate cultures. Immunoglobulin values were obtained by interpolation from standard curves. Statistical analysis Data were analysed using "Student's t-test". Probability values below 5% were considered significant. Results EBV serology All patients showed a typical serological profile characteristic of NPC as determined by indirect immunofluorescence. Levels of IgG antibodies to VCA were considerably higher than those in healthy donors. Their titers varied from 640 to 2560 (mean titer = 1065), whereas in healthy donors such titers fluctuated between 40 and 160 (mean titer = 70). IgG antibodies to EA were present in NPC patients only, with titers ranging from 20 to 320 (mean titer = 50). Anti-VCA IgA antibodies were detected in all patients (range : 20–320 ; mean = 61) and anti-EA IgA antibodies were detected in only 4 patients (range : 10–80 ; mean = 27). None of the controls showed IgA antibodies against these antigens. Immunophenotyping of lymphocyte preparations Immunophenotypic analysis of the lymphocyte preparations obtained from patients and controls indicated that CD3+ lymphocytes constituted the major subpopulation with a mean frequency of (60 ± 8)% in PBL of both patients and controls and (55 ± 9)% in TIL. CD4+ lymphocytes represented (35 ± 5)% in PBL of both patients and controls and (20 ± 3)% in TIL. CD8+ cells showed mean values of (40 ± 6)% in PBL of patients, (36 ± 4)% in TIL, and (28 ± 5)% in PBL of controls. On the other hand, CD19+ B lymphocytes represented in average (25 ± 5)% in PBL of patients, (23 ± 6)% in TIL, and (22 ± 3)% in PBL of controls. Cytokine production As illustrated in Figures 1 and 2 , the PBL of NPC patients produced significantly more IL-2 (2105 ± 1152 pg/ml) and IL-10 (1280 ± 727 pg/ml) than the PBL of healthy donors (1286 ± 452 pg/ml, p = 0,022 for IL-2 and 793 ± 325 pg/ml, p = 0,016 for IL-10). Comparable amounts of IL-6 were found for both groups (2375 ± 919 pg/ml for patients and 2177 ± 435 pg/ml for controls, p = 0,429 ; Figure 3 ). On the other hand, TIL of patients showed a significantly higher IL-2 (3913 ± 1484 pg/ml, p = 0,0002) and IL-10 (1926 ± 817 pg/ml, p = 0,02) production than their PBL. The observed differences in mean IL-6 production between patients'PBL (2375 ± 919 pg/ml) and TIL (1962 ± 515 pg/ml) did not reach statistical significance (p = 0,116). Unstimulated lymphocytes cultured in the absence of PWM did not show any detectable cytokine production (data not shown). Immunoglobulin production The results of immunoglobulin determination showed that the PBL of NPC patients produced significantly higher levels of IgM (8262 ± 5315 ng/ml) than the PBL of controls (3753 ± 2801 ng/ml, p = 0,004 ; Figure 4 ). Both groups produced similar amounts of IgG (874 ± 408 ng/ml for patients and 847 ± 442 ng/ml for controls, p = 0,85 ; Figure 5 ) and IgA (4380 ± 4316 ng/ml for patients and 3067 ± 2267 ng/ml for controls, p = 0,27 ; Figure 6 ). On the other hand, TIL of these patients produced significantly higher levels of IgM (21162 ± 12276 ng/ml, p = 0,0003) and IgG (2789 ± 1583 ng/ml, p < 0,0001) than their PBL. No significant differences in IgA production were found between PBL (4380 ± 4316 ng/ml) and TIL (6112 ± 6046 ng/ml, p = 0,34). Unstimulated lymphocytes cultured in the absence of PWM did not show any detectable immunoglobulin production (data not shown). Discussion Previous reports indicate the absence of EBV-specific cytotoxic T lymphocytes detectable by the standard chromium release assay in both the peripheral and intratumoral compartments [ 8 - 10 , 21 ] in undifferentiated nasopharyngeal carcinoma. However, NPC patients show a strong EBV-specific humoral immune response, and elevated titers of anti-EBV antibodies directed against viral antigens are observed in their sera [ 11 , 12 , 24 ]. The mechanisms underlying this discrepancy between humoral and cellular immune responses in NPC patients are still unknown. It was postulated that interleukin 10 production in malignant tumors would facilitate their escape from immune surveillance [ 16 ]. The expression of IL-10 in NPC has been controversial. While some authors have reported that NPC tumor cells do not express IL-10 [ 17 ], others observed IL-10 expression in epithelial NPC tumor cells and tumor infiltrating lymphocytes [ 18 , 19 ]. They suggested such IL-10 expression as a possible mechanism for NPC tumors and EBV to escape local cellular immune attack. Indeed, IL-10 is a pleiotropic factor known for its suppressive effects on cell-mediated immune responses [ 22 ]. In sharp contrast to these inhibitory effects, IL-10 also has a potent stimulatory effect on the humoral immune response, inducing B lymphocyte differentiation and immunoglobulin secretion [ 23 ]. Therefore, IL-10 hyperproduction alone or in association with changes in other cytokines might lead to an imbalance between humoral and cellular immune responses similar to that seen in NPC. The expression of cytokines such as IL-2 [ 19 ], IL-6 [ 17 ] and IL-10 [ 18 , 19 ] in NPC has been studied mainly on tumor biopsies using immunohistochemical and molecular techniques. To the best of our knowledge, only one study on the ability of NPC patients'lymphocytes to produce cytokines in culture has been reported and it was limited to IL-2 [ 9 ]. In this report, we investigated the ability of both peripheral blood and tumor infiltrating lymphocytes of 17 undifferentiated NPC patients to produce cytokines following mitogenic stimulation in culture. Since immunoglobulin isotypes are determined by cytokine patterns, we also looked for possible correlations between measured cytokine levels and immunoglobulin isotypes produced. PWM was chosen to stimulate the lymphocyte cultures because it is known to activate both T and B lymphocytes in humans [ 29 , 30 ]. In addition, the ability of PWM to stimulate cytokine production by Tcells is similar to that of PHA [ 31 ]. This allowed us to study the responses of both T and B cells simultaneously in each lymphocyte culture. The data obtained indicate that the highest levels of IL-2 were produced by TIL cultures followed by patients'PBL. This is in line with a report by Lakhdar et al [ 9 ] showing a higher IL-2 production by PHA-stimulated PBL of NPC patients than by controls. Such high IL-2 levels are expected to favor a strong cytotoxic response since IL-2 is needed for CTL stimulation and proliferation [ 32 ] and they are not consistent with the known lack of detectable CTL activity. IL-10, a representative of the Th2 pattern of cytokines, is generally considered immunosuppressive. It inhibits IL-6 secretion by activated macrophages but not by Th2 lymphocytes [ 33 ]. In the present work, IL-10 was overproduced by patients'lymphocyte cultures whereas their IL-6 levels were similar to controls, showing no signs of inhibition by IL-10. This points to Th2 lymphocytes rather than activated macrophages as the main source of IL-6 in this system. The increased ability of patients lymphocytes to produce IL-10 is compatible with the lack of CTL activity. Cultures of tumor infiltrating lymphocytes secreted significantly higher amounts of IgM and IgG than the PBL of either patients or controls in good agreement with the increased levels of IL-2 and IL-10, since these cytokines are involved in B cell activation and enhance immunoglobulin synthesis [ 23 , 25 ]. No significant correlations between cytokine production and IgA secretion were found, in line with the preferential enhancing effect of IL-2 and IL-10 on IgM and IgG synthesis [ 23 ]. In an attempt to see whether the observed differences in cytokine and immunoglobulin production between patients and controls or between PBL and TIL of patients could be due to differences in the composition of individual lymphocyte preparations, we performed an immunophenotypic analysis of each lymphocyte preparation. As shown in the results, the only significant changes in the proportions of lymphocyte subsets between patients and controls were observed in the CD8 subpopulation which showed an increase in patients PBL and TIL. On the other hand, the CD4 subset showed a significant decrease in TIL. Such differences in lymphocyte subpopulations are not expected to produce the changes in cytokine and immunoglobulin production observed here, since CD4 lymphocytes are well known for being the main source of IL-2 and IL-10, and for their ability to stimulate immunoglobulin synthesis [ 34 ]. Recently, CD4+ lymphocyte populations have been shown to be more heterogeneous and complex than previously thought [ 35 ] and new subsets of T regulatory cells (Tr) with immunosuppressive activities have been identified. Tr cells have been suggested to play a key role in the evasion from immune-mediated clearance of microorganisms and tumors [ 36 ]. It has also been reported that IL-10-producing Tr1 cells dominate the immune response to LMP1 in EBV seropositive subjects [ 36 ]. Since LMP1 is expressed in NPC tumors, it would be tempting to speculate that the increase in IL-10 production by TIL would correspond to an increase in Tr1 cells in the corresponding NPC tumors. In this respect, Hodgkin lymphoma infiltrating lymphocytes have been shown to contain large populations of both Tr1 and CD4+CD25+ regulatory T cells [ 37 ], and it would be interesting to see whether a similar situation occurs in NPC. Conclusion In conclusion, our data point to the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. Their lymphocytes, especially those infiltrating the tumors, showed in particular a high propensity to produce IL-10 following mitogenic stimulation in vitro. Overproduction of this pleiotropic cytokine may lead to a discrepancy between humoral and cellular immune responses similar to that seen in NPC. The relevance of the present observations to the in vivo situation is not yet established and warrants further investigations. Competing interests The authors declare that they have no competing interests. Authors' contributions LFJ designed and carried out the experimental work, performed the statistical analysis and drafted the manuscript. NK maintained the cell cultures and provided technical help. RHK and KB participated in the design and coordination of the study. RK directed the research and finalized the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522818.xml |
552333 | Haematuria as a presentation of metastatic oesophageal carcinoma | Haematuria is a classical symptom of urological disease often signifying a primary bladder cancer. Rarely, however, the presence of blood in the urine can be due to secondary spread of tumours into the bladder from distant sites. Notably this has been reported to occur in breast cancer, malignant melanoma and gastric cancers. Haematuria due to spread from a primary oesophageal cancer to the bladder has never been reported. We present a case of haematuria confirmed histologically to be due to metastases from a primary oesophageal tumour. Oesophageal cancer is capable of spread to all three neighbouring compartments (abdomen, chest and neck) and therefore has the potential to spread to unusual sites. Clinicians should always carefully regard haematuria in a patient previously treated for cancer and retain a high index of suspicion for distant metastases as being the cause. | Background Haematuria is a commonly encountered symptom. It often represents the presence of serious disease such as a malignancy within the bladder. The majority of bladder tumours tend to be primary, and histologically these are usually transitional cell carcinomas. We present a case of haematuria which occurred due to metastases from a primary oesophageal carcinoma diagnosed 2 years prior and treated curatively. Case presentation A 45-year old male presented to our unit with acute onset macroscopic haematuria. His past medical history was significant in that he had been diagnosed with adenocarcinoma of the distal oesophagus 2 years prior and had undergone curative resection after neo-adjuvant chemotherapy. At that time the tumour was found to be poorly differentiated with evidence of local nodal spread. He had been reviewed regularly by the oncologists and remained asymptomatic until the onset of frank haematuria. He subsequently underwent cystoscopy, which revealed a solid bladder tumour on the right lateral wall, which was treated with trans-urethral resection. Pathological examination confirmed a poorly differentiated mucus-secreting adenocarcinoma, identical histologically to the original oesophageal tumour (Figures 1 and 2 ). Figure 1 Original oesophageal adenocarcinoma (H & E stain). Figure 2 Metastatic tumour to the bladder (H & E stain). Both light micrographs demonstrate extensive infiltration by a poorly differentiated adenocarcinoma with the same histopathological features. A diagnosis of metastatic oesophageal adenocarcinoma to the bladder was made. A CT scan did not demonstrate any pelvic tumour outside the bladder and therefore metastasis by the trans-coelomic route was essentially excluded, indicating haematogenous spread of the primary oesophageal carcinoma. The patient was referred for further oncological therapy but unfortunately died 4 months later from disseminated carcinoma. Discussion Metastatic tumour spread to the bladder constitutes approximately 2% of all bladder neoplasms [ 1 ]. Gross haematuria occurs relatively infrequently in secondary tumours of the bladder as most lesions are small and infiltrate the bladder wall without causing ulceration of the mucosa [ 2 ]. Therefore most metastases to the bladder remain asymptomatic and often undiagnosed. The bladder can be the recipient of metastatic tumour spread from a potentially large variety of primary sites. Most commonly direct invasion can occur from adjacent tumours of the lower gastrointestinal tract (33% of secondary neoplasms), prostate (19%) and female genital tract (11%) [ 1 ]. Less commonly, distant metastases have been described, notably from the stomach, skin, breast and lung in descending order of frequency [ 2 - 5 ]. The management and prognosis of such tumours can differ significantly from that of primary bladder tumours since they are often indicative of late disease. Despite curative intent, surgical resection of oesophageal adenocarcinoma is associated with an overall tumour recurrence rate of 66% at 5 years [ 6 ]. The lymphatic drainage of the oesophagus is longitudinal via the submucosal plexus and not segmental. As a consequence lymph node metastases can occur relatively early in all three compartments (abdomen, chest and neck) regardless of the location of the primary tumour [ 7 ]. In autopsy studies, isolated lymph node metastases were found in approximately one half of patients with end-stage oesophageal carcinoma, with a similar proportion having combined lymph node and visceral metastases. Isolated visceral spread however is rare, accounting for only a handful of cases of primary oesophageal tumour spread [ 8 ]. Notable sites of haematogenous dissemination of primary oesophageal carcinoma to distant organs include bone, liver, skin, lungs, adrenals, brain and peritoneum in descending order of frequency [ 6 ]. A few authors, most notably in Japan, have described cases of oesophageal cancers metastasising to the kidney. These cases may present with haematuria but often are associated with flank pain [ 9 - 14 ]. Interestingly, rare synchronous primary tumours of the bladder and oesophagus have been described [ 15 , 16 ] but haematuria due to secondary spread of oesophageal cancer has never previously been reported. Conclusion Haematuria may be the only clinically apparent symptom of metastatic tumour spread to the bladder from a potentially large number of primary sites and therefore should be considered by all clinicians irrespective of specialty. Competing interests The author(s) declare that they have no competing interests. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552333.xml |
553993 | Correlation of exhaled breath temperature with bronchial blood flow in asthma | In asthma elevated rates of exhaled breath temperature changes (Δe°T) and bronchial blood flow (Q aw ) may be due to increased vascularity of the airway mucosa as a result of inflammation. We investigated the relationship of Δe°T with Q aw and airway inflammation as assessed by exhaled nitric oxide (NO). We also studied the anti-inflammatory and vasoactive effects of inhaled corticosteroid and β 2 -agonist. Δe°T was confirmed to be elevated (7.27 ± 0.6 Δ°C/s) in 19 asthmatic subjects (mean age ± SEM, 40 ± 6 yr; 6 male, FEV 1 74 ± 6 % predicted) compared to 16 normal volunteers (4.23 ± 0.41 Δ°C/s, p < 0.01) (30 ± 2 yr) and was significantly increased after salbutamol inhalation in normal subjects (7.8 ± 0.6 Δ°C/ s, p < 0.05) but not in asthmatic patients. Q aw , measured using an acetylene dilution method was also elevated in patients with asthma compared to normal subjects (49.47 ± 2.06 and 31.56 ± 1.6 μl/ml/min p < 0.01) and correlated with exhaled NO (r = 0.57, p < 0.05) and Δe°T (r = 0.525, p < 0.05). In asthma patients, Q aw was reduced 30 minutes after the inhalation of budesonide 400 μg (21.0 ± 2.3 μl/ml/min, p < 0.05) but was not affected by salbutamol. Δe°T correlates with Q aw and exhaled NO in asthmatic patients and therefore may reflect airway inflammation, as confirmed by the rapid response to steroids. | Asthma is an inflammatory disease of the airways. Vasodilatation is a critical feature of inflammation, and angiogenesis and vascular remodelling are features of chronic inflammatory diseases, such as asthma [ 1 ]. The increased vascularity of the airways in asthma [ 2 ] is partly due to the elevated number of vessels associated with angiogenesis and partly due to vasodilation caused by the release of vasodilator mediators, such as, histamine, bradykinin [ 3 ], and nitric oxide (NO) [ 4 ]. In a recent study we have found that patients with asthma have higher increases of exhaled breath temperature (Δe°T) compared with normal subjects and that this is correlated to the concentration of exhaled nitric oxide (NO) [ 5 ]. Therefore, we suggested that patients with asthma have high Δe°T. and that this may be due to airway inflammation and elevated levels of NO. In the present study we hypothesise that elevated Δe°T may result from increased heat exchange in the airways due to elevated bronchial blood flow (Q aw ) caused by inflammation and airway remodelling. To test this, we investigate the relationship between airway inflammation as assessed by exhaled NO, with Q aw and Δe°T measured non-invasively. Q aw is an expression of bronchial blood flow, whereas Δe°T reflects the rate of temperature increase in the exhaled breath. We hypothesised that Q aw changes may contribute to the levels of Δe°T and we studied their relationship considering that, potentially, minor changes of bronchial blood flow may not affect exhaled breath temperature. Elevated levels of exhaled NO in asthma [ 6 , 7 ] are likely to be due to the activation of the inducible form of NO synthase (iNOS) by inflammatory cytokines [ 8 ] and therefore, reflect airway inflammation. Because NO also regulates bronchial vascular tone [ 9 ] and may increase bronchial blood flow [ 10 , 11 ] we measured its concentration in the exhaled breath as a marker of inflammation and we analysed its relationship with Q aw and Δe°T. The measurement of Q aw is non-invasive and was standardised and adapted from a previously validated technique [ 12 , 13 ]. Δe°T and bronchial blood flow (Q aw ) were also measured non-invasively allowing us to make repeated measurements and to study the interactions of these two markers and exhaled NO. The inhalation of corticosteroids has been shown to have an acute vasocostrictive effect on the bronchial circulation [ 14 ] and the measurement of Q aw has been advocated to assess airway steroid sensitivity. In order to evaluate further the correlation of Q aw and Δe°T with inflammation, we studied the acute effect of inhaled budesonide on these parameters and their reciprocal changes. Furthermore, to validate our methods, we also evaluated the vasodilating effect of the short-acting β 2 -agonist salbutamol. Methods Patients Nineteen asthmatic patients were studied (7 male, age 40 ± 5 yr, FEV 1 74 ± 6 %, 9 patients were on inhaled steroid treatment and 10 patients had mild persistent asthma and were on β 2 -agonist inhalers only). These two groups of patients were chosen because they are representative of the larger majority of asthmatic patients; in addition this allowed us to verify cross sectionally the effect of inhaled steroids on bronchial blood flow and exhaled NO. We also examined 16 control subjects (age 30 ± 2 yr, 8 male) recruited from our outpatient clinic and from volunteers (Table 1 ). Most of these subjects had previously taken part in other published studies. The diagnosis of asthma was established in each patient according to American Thoracic Society criteria [ 15 ]. Patients with acute chest infection or disease exacerbation during the month before enrolment were excluded. Patients with history of diabetes, liver disease, heart failure, lung cancer, or alcohol/drug abuse were not eligible for the study. All subjects were life-long non-smokers. All asthmatic patients refrained from using β 2 agonists and corticosteroids for at least 12 h prior to the study. The tests were carried out at ambient temperature between 23 and 25C° and humidity 60% and 65%. Table 1 PATIENT CHARACTERISTICS Asthma not steroid treated (n = 10) Steroid treated asthma (n = 9) Normals (n = 16) Age (years) 37 ± 7 45 ± 7 30 ± 2 Sex (M/F) 4/6 3/6 8/8 FEV 1 (% predicted) 93 ± 10 75 ± 11 97 ± 9 Smokers 0 0 0 Ex smokers 0 0 0 Therapy: Inhaled β-drenergics 10 10 0 Theophylline 0 0 0 Inhaled steroids 0 10 0 Oral steroids 0 0 0 Definition of abbreviations: ; FEV 1 = forced expiratory volume in one second. Values are means ± SEM. Study design The study was approved by the Brompton and Harefield NHS trust Ethics Committee. After a clinical examination was carried out, Δe°T, Q aw and exhaled NO were measured at least after one hour of rest in the laboratory. This was followed by spirometry. Eight asthmatic subjects and eight normal volunteers agreed to have Δe°T and Q aw measured after the inhalation of budesonide 400 μg and salbutamol 200 μg or placebo, which were administered in three different visits. The measurements were repeated every 15 minutes for 1 h after budesonide and placebo inhalation and at baseline and every 10 minutes for half an hour after the inhalation of salbutamol. Exhaled breath temperature measurement During a flow and pressure controlled single breath exhalation exhaled breath temperature gradients were measured as previously described [ 5 ]. As previously shown [ 5 ], during a flow and pressure controlled exhalation from total lung capacity through a 2.77 mm mouthpiece[ 16 ] exhaled breath temperature was measured by a fast response (1 ms) high accuracy (0.015 ± 0.027°C), thermometer (Picotech Ltd) interfaced with a computer by a single channel Picotech Oscilloscope (model ADC 42, resolution 12 bits) allowing online recording of exhaled breath temperature. In a preliminary study exhaled breath temperature tracings were analyzed mathematically. The tracings proved to have an exponential rise and the point at 63% of the total temperature increase was chosen to study the slope of the curves because it represents two time constants of the maximal °T change and therefore allows a better mathematical characterization of the tracings before plateau. The time constant of the thermometer response was found by measuring the time for the temperature to reach 63% of the final reading. This avoided large errors in estimating when the asymptotic final reading had been reached. The exhaled breath temperature changes exponentially with time. The shape of the curve depends upon the time constant (T). In one time constant the response reaches 63% of its final change. The response is of the form Where V 2 is the final value, V 1 the initial value, and t is the time after exhalation was started. As t > infinity, the exponential term > 0. Hence the response rises asymptotically to its final value. When performing experiments to determine the time response of a system, it is difficult to tell at which time the final value is obtained, since there will be a small change in the response for a relatively long time as the asymptote is approached. Jitter and noise in the signal will also add to this problem. It is easier to estimate the asymptotic value and find the time at which a certain percentage of this is reached. Various percentages are possible, the most commonly used in physics and biology is the time constant , although in some applications (particularly electronic engineering) the rise time to 95% of the final value is used. The 63% arises because when t = T, the expression above becomes ( V 2 - V 1 )(1-1/e) = ( V 2 - V 1 )(1-0.37) = 0.63( V 2 - V 1 ) In n time constants the percentage reached is 100 × (1-1/e n ) which, approaches 98% after about 5 time constants. The rate of temperature increase (Δe°T) calculated between the beginning of exhalation and 63 % of the total temperature increase (a/b, where " a " is 63% of Δ°T and " b " the time to reach " a ") proved to be the more reproducible parameter to characterize the curves. We evaluated the effect of different exhalation flow rates, distance of the thermocouple from the edge of the mouthpiece and ambient temperature on Δe°T and end-expiratory plateau temperatures. We found that Δe°T but not plateau temperatures were elevated at low (2–3 L/min) compared to higher (5–6 L/min) exhalation flow rates (6.25 ± 0.4°C/s and 4.45 ± 0.8°C/s, p < 0.01) in 8 normal subjects. When the thermocouple was inserted close to the edge of the mouthpiece (1 cm) the Δe°T was significantly higher (7.15 ± 0.2°C/s) compared to when it was located farther (2 cm) (4.45 ± 0.8°C/s, p < 0.01). There was a tendency for faster Δe°T when the subjects were starting exhaling from higher baseline ambient temperatures but this was not significant for temperature changes within ± 3°C (5.05 ± 0.8°C/s at 28°C and 4.45 ± 0.8°C/s at 22°C p > 0.05). The volume ventilation did not influence the Δe°T value. The difference in exhaled breath Δe°T and plateau temperatures measured during two successive collections at five minutes intervals (single session variability) was 4.4%, while between sessions variability (n = 6, one day interval) was 6.8%. The reproducibility of the test was confirmed by the Bland and Altman test [ 17 ]. Exhaled NO measurements Exhaled NO was measured using a modified chemiluminescence analyzer (model LR2000; Logan Research, Rochester, Kent) as previously described [ 18 ]. Bronchial blood flow We modified a previously validated soluble inert gas uptake method to measure Q aw , using acetylene rather than the potentially explosive dimethylether [ 12 , 13 ]. The subjects were sitting in front of a valve system inhaling through a mouthpiece (with nose clips on) initially room air and then a gas mixture from a Teflon bag containing 35% O2, 0,3% acetylene, 5% sulphur hexafluoride, CO 3% and a balance of nitrogen. During the exhalation the concentration of acetylene was measured directly online by a mass spectrometer, and Q aw was calculated from the Fick principle (dilution of acetylene concentration) (Figure 1 ), the area under the curve (AUC) being inversely proportional to the bronchial blood flow. Q aw was expressed as μl/ml/min representing the volume of blood per volume of dead space per time. Figure 1 Method for the measurement of bronchial blood flow (Q aw ). Subjects inhale 60% of their vital capacity from a reservoir containing acetylene 0.3% and then exhale into a mass spectrometer (Panel A). Panel B shows a tracing of acetylene, the area under the curve, corresponding to the conducting airways, is proportional to airway blood flow. Previous studies have used dimethyl ether instead of acetylene for the measurement of bronchial blood flow. In the current study we used acetylene because of the higher explosive potential of dimethylether when in contact with O 2 . This method was previously validated in the sheep where the invasive inoculation of radioactive micro spheres directly into the bronchial circulation provided similar results [ 19 ]. The use of micro spheres is considered the Gold Standard for the measurement of blood flow, however, this method is invasive and presents limitations such as the recirculation of the radioactive spheres. Acetylene and diethyl ether present similar blood solubility and affinity for haemoglobin when measured at the same temperature [ 20 ]. Gas exchange efficiency is largely dependent on solubility. Because these two gases have similar physicochemical characteristics we assume that they can be used interchangeably. This is further confirmed by the finding of a similar range of bronchial blood flow and similar response to corticosteroids (vasoconstriction) and beta 2 agonists (vasodilatation) in this study compared to previously published studies which used the dimethyl ether method. Statistics Comparisons between groups were made by one-way analysis of variance (ANOVA).. Data were expressed as means ± standard error of mean. The relationship between the exhaled breath temperature, Q aw, NO and FEV 1 were tested with the linear correlation coefficient. Results Bronchial blood flow (Q aw ) Q aw was elevated to a similar extent in patients with mild persistent and moderate asthma (on regular inhaled corticosteroids and β 2 -agonists) (46.0 ± 51 μl/ml/min) and patients with mild intermittent asthma (on β 2 -agonists as needed only) (52.64 ± 3.0 μl/ml/min, p > 0.05) compared to normal subjects (31.56 ± 1.6 μl/ml/min, p < 0.01, Figure 2 , Panel A). Q aw was correlated with exhaled NO (r = 0.57, p < 0.05, Figure 3 , Panel A) and Δe°T (r = 0.52, p < 0.05, Figure 3 , Panel B). Figure 2 Levels of bronchial blood flow (Q aw ) (Panel A) and exhaled breath temperature gradients (Δe°T) (Panel B) in normal subjects (□) and patients with asthma (•). Figure 3 Correlation of bronchial blood flow (Q aw ) with exhaled nitric oxide (NO) (Panel A) and exhaled breath temperature gradients (Δe°T) (Panel B) in patients with asthma. Exhaled air temperature Δe°T was higher in asthmatic patients (7.27 ± 0.6 Δ°C/ sec) compared to normal subjects (4.23 ± 0.41 Δ°C/ sec, p < 0.01, Figure 2 , Panel B) and was not statistically different in steroid treated (7.56 ± 0.99 Δ°C/ sec) compared to untreated patients (6.83 ± 0.78 Δ°C/ sec, p > 0.05). Exhaled NO NO levels were elevated in asthmatic subjects not on steroid treatment (15.6 ± 2.8 ppb) compared to steroid treated patients (7.5.6 ± 2.3 ppb, p < 0.05) and normal subjects (4.7 ± 0.3 ppb, p < 0.05). Effect of budesonide and salbutamol inhalation Bronchial blood flow In asthmatic patients Q aw was significantly reduced 30 minutes after the inhalation of budesonide compared to baseline (53.0 ± 5.0 μl/ml/min and 21.3 ± 2.32 μl/ml/min respectively, p < 0.05 Figure 4 Panel A) and returned to baseline levels at 60 minutes (52.6 ± 4.0 μl/ml/min). In normal subjects there was a tendency for lower Q aw after the inhalation of budesonide but such changes were not significant (30.3 ± 5.0 μl/ml/min and 26.3 ± 3.0 μl/ml/min at baseline and at 30 minutes respectively, p > 0.05, n = 5, Figure 4 Panel A). Figure 4 Acute effect of budesonide inhalation (400 μg) (Panel A) and salbutamol (200 μg) (Panel B) on bronchial blood flow (Q aw ). In 8 normal volunteers Q aw was increased after salbutamol inhalation (32.3 ± 8.1 μl/ml/min and 55.0 ± 3.0 μl/ml/min at baseline and at 10 minutes respectively, p < 0.05), while this effect was not present in asthmatic patients (54.0 ± 7.0 μl/ml/min and 52.0 ± 6.0 μl/ml/min, p > 0.05, n = 5, Figure 4 Panel B). Placebo had no effect on Q aw in subjects with asthma (54.0 ± 3.0, 56.1 ± 4.5 and 53.1 ± 8.1 μl/min/ml/ at baseline, 30 and 60 minutes respectively, p > 0.05) nor in normal subjects (34.0 ± 3.0,38.1 ± 4.5 and 35.1 ± 8.1 μl/min/ ml/ p > 0.05) Exhaled breath temperature The inhalation of budesonide was associated with a tendency for a decrease of Δe°T in asthmatic patients (from 10.17 ± 2 Δ°C/ sec at baseline to 8.6 ± 3 Δ°C/sec 30 minutes after inhalation Figure 5 Panel A), even though this decrease was not significant; the changes in Q aw and Δe°T were correlated in asthmatic patients (r = 0.78, p < 0.05). In normal subjects, no significant changes of Δe°T were found at any of the time points after budesonide inhalation (3.5 ± 2 Δ°C/sc at baseline, 3.5 ± 1 Δ°C/sc at 30 minutes, 3.53 ± 2 Δ°C/sc at 60 minutes, p > 0.05). Figure 5 Acute effect of budesonide inhalation (400 μg) (Panel A) and salbutamol (200 μg) (Panel B) on exhaled breath temperature gradients (Δe°T). In 5 normal volunteers Δe°T was increased after the inhalation of 200 μg of salbutamol (3.50 ± 0.29 Δ°C/ sec and 7.8 ± 0.6 Δ°C/ sec, p < 0.01), while this effect was not present in asthmatic patients (10.1 ± 0.44 Δ°C/ sec and 9.67 ± 0.51 Δ°C/ sec, p > 0.05, n = 5, Figure 5 Panel B) Δe°T was unchanged in subjects with asthma (9.17 ± 2 and 10.23 ± 3.5 Δ°C/ sec at baseline and 10 minutes respectively, p > 0.05) and in normal subjects (3.50 ± 0.29 Δ°C/ sec and 4.20 ± 0.32 Δ°C/ sec p > 0.05) after the inhalation of placebo. Discussion This is the first study to show that elevated levels of Q aw and Δe°T are correlated with one another and with airway inflammation as assessed by exhaled NO. We propose that high levels of NO generated in asthmatic patients as a result of airway inflammation may cause vasodilatation of the bronchial circulation contributing to increased heat exchange. This is supported by the demonstration that lower Δe°T levels, after the inhalation of corticosteroids, are correlated with reduced levels of bronchial blood flow. We propose that these non-invasive measurements may be useful to evaluate airway inflammation and may provide a tool to assess steroid sensitivity. Angiogenesis and microvascular remodelling are features of chronic inflammatory diseases, such as asthma [ 21 ]. As inflammatory diseases evolve, the microvasculature undergoes progressive changes in structure and function. Blood vessels enlarge and proliferate supplying inflammatory cells in chronically inflamed tissues. Because of these changes, asthmatic patients have increased vascularity of the airway mucosa which is related to the severity of the disease [ 2 ]. Airway vascular remodelling and inflammation maybe responsible for increased bronchial blood flow [ 22 ] and exhaled breath temperature gradients in asthmatic patients [ 5 ]. In a previous study [ 23 ] we have proved that patients with asthma have elevated Δe°T compared to normal subjects and because we found a significant correlation with exhaled NO we suggested that this was due to airway inflammation. In the present study we hypothesised that Δe°T is elevated in asthma as a result of increased bronchial blood flow and we studied the relationship between Q aw and Δe°T and airway inflammation as assessed by exhaled NO. Even though the patients enrolled in this study were significantly older than the control group, our previous studies [ 5 , 24 ] indicate that that age does not affect Δe°T. We studied airway inflammation measuring exhaled NO and we investigates its relationship with Q aw which has also been suggested as a marker of inflammation. We also studied the interaction of these two parameters after inhaled corticosteroids β 2 agonists. We confirmed previous data showing elevated levels of Q aw in asthmatic subjects compared to normal volunteers, using a modified method developed by Onorato et al [ 25 ]. In the current study we preferred the use of acetylene over dimethyl ether because the latter is highly explosive when in contact with oxygen. Gas exchange efficiency is largely dependent on solubility, because these two gases have similar physicochemical characteristics we assume that they can be used interchangeably. Furthermore, the measurement of Q aw in this study presented the same response pattern and timing to the inahaltion of steroids [ 12 ] and beta agonists [ 26 ] as previously showed using the dimethyl ether method confirming that the method presented in our study is an acceptable measurement of Q aw . The method used in this study for the measurement of Q aw produces results which include the contribution of the dead space and trachea blood flow to the total bronchial blood flow. We acknowledge that the trachea may not be the main site of inflammation in asthma, but inflammation in asthma extends from the larynx to the terminal bronchioles and the tracheal mucosa certainly appears inflamed in many asthmatic patients. When tracheal inflammation occurs there will be a good separation between normal subjects and patients with asthma because the measurements of bronchial blood flow and exhaled breath temperature will particularly reflect the contribution of this part of the respiratory tract For the first time we have shown, using non-invasive methods, that changes in bronchial blood flow can alter exhaled breath temperature indicating that the bronchial circulation may control airstream temperature. Furthermore, in this study not only we have shown a correlation between Q aw and Δe°T but we have also shown that these measurements respond similarly to steroids and beta 2 agonists. Therefore, patients with asthma have a significantly faster rise of breath temperature and Q aw and these are correlated. We presume that this is due to the increased vascularity of the bronchial vessels [ 27 ] and elevated blood supply and therefore heat transfer across the bronchial wall. Hyperaemia and hyperperfusion are consistent features of tissue inflammation, therefore, the finding of increased exhaled air temperature and bronchial blood flow in asthmatic patients may be due to the elevated levels of exhaled NO which is a marker of inflammation and a potent bronchial vasodilator. Even though the correlation between Q aw and Δe°T may appear to be weak, it is noteworthy that the acute changes of these variable was significantly correlated, reinforcing the hypothesis that elevated breath temperature gradients in asthmatic patients may reflect increased bronchial blood flows. We were able to show a positive correlation between exhaled NO and Δe°T. NO is a gas produced by several types of pulmonary cells, including inflammatory, endothelial and airway epithelial cells. Elevated levels of exhaled NO in asthma [ 6 ], and interstitial lung disease [ 28 ] are likely to be due to the activation of the inducible form of NO synthase (iNOS) and therefore may reflect airway inflammation, alternatively NO maybe produced by the bronchial epithelium. In addition the activity of iNOS, the inducible enzyme responsible for the synthesis of NO, is temperature-dependent [ 29 ], therefore elevated airway temperatures in patients with asthma [ 5 ] may induce further synthesis of NO. NO is a potent vasodilator and may play a role in the regulation of bronchial vasomotor tone [ 10 ], so that elevated levels of NO may lead to vasodilatation and increased bronchial blood flow as shown by the correlation between exhaled NO and Δe°T. Unfortunately, in the current study, we were unable to establish the contribution of NO produced by the bronchial vasculature compared to the pulmonary circulation. In this cross-sectional study we could not show any differences of Δe°T or Q aw in corticosteroid-treated compared to untreated asthmatic patients, despite the efficacy of steroids in reducing bronchial blood flow [ 12 ]. This is consistent with previous studies published by our group and others showing that steroid-treated asthmatic patients have similar Δe°T [ 5 ] and Q aw [ 22 ] compared to untreated patients. One hypothesis is that the vasoconstrictive action of inhaled corticosteroids may have been balanced by β 2 induced vasodilatation resulting in minimal changes in bronchial artery diameter and blood flow and therefore no changes of Q aw and Δe°T. However, these results must be confirmed by placebo controlled studies. In contrast to the effect of chronic treatment with corticosteroids, the acute inhalation of budesonide caused a significant temporary reduction of Q aw which returned to baseline one hour after inhalation. This is also consistent with a previous publication [ 22 ], notably, in the current study Q aw and Δe°T and their interaction were studied simultaneously in the same group of patients for the first time. Corticosteroids may cause vasoconstriction by numerous mechanisms. They can potentiate the vasoconstrictor actions of noradrenalin and angiotensin II by upregulating their vascular receptors [ 30 ]. Furthermore, corticosteroids may inhibit the synthesis of NO [ 31 ], thus causing vasoconstriction. In addition to these mechanisms of action, corticosteroids may also have a very rapid action (less than 5 minutes) by inhibiting noradrenalin uptake in bronchial blood vessels [ 32 ]. The glucocorticoid-induced vasoconstriction in asthmatics seems to be accompanied by a greater α 1 -adrenergic vasoconstrictor response [ 26 ]. This adds further support to a α 1 -adrenergic steroid interplay in the regulation of vascular tone. Further studies are required, investigating the dose response relationship for inhaled steroids would provide valuable information. The cardinal signs of inflammation are rubor (redness), calor (heat), tumor (swelling), dolor (pain), and impaired function (functio laesa). Exhaled breath temperature and bronchial blood flow may reflect rubor and calor in the airways and therefore may be markers of tissue inflammation and remodelling as confirmed by the positive correlation between Δe°T, Q aw and exhaled NO which was shown for the first time in this study. Measurement of exhaled breath temperature and bronchial blood flow may provide means of detecting airway inflammation and vascular remodelling in a non-invasive way. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553993.xml |
549061 | Global expression changes resulting from loss of telomeric DNA in fission yeast | Gene expression profiling of the response of Schizosaccharomyces pombe cells to loss of the catalytic subunit of telomerase ( trt1 + ) identified two waves of altered gene expression and a continued up-regulation of Core Environmental stress Response (CESR) genes. | Background Telomeres are the nucleoprotein ends of linear eukaryotic chromosomes. In most organisms, telomeric DNA consists of a simple, repeated sequence with a G-rich strand running 5' to 3' towards the chromosome end, and terminates with a short, single-stranded 3' overhang (reviewed in [ 1 , 2 ]). The length of the duplex repeated region varies, from 20 base-pairs (bp) in hypotrichous ciliated protozoa to around 300 bp in yeast and several kilobases (kb) in mammalian cells. These DNA repeats recruit telomeric proteins to form the telosome, a structure that resists nucleolytic degradation and prevents chromosome ends from eliciting recombination and end-joining pathways for repairing double-strand DNA breaks [ 3 ]. Telomeres are also essential for the complete replication of chromosomes, because conventional DNA polymerases do not copy the extreme ends of linear DNA molecules. In the absence of a mechanism to compensate for this 'end-replication problem', progressive telomere shortening leads to replicative senescence, which in yeast is characterized by chromosome instability and low cell viability [ 4 , 5 ]. Replicative senescence in mammals is characterized by growth arrest and altered gene expression [ 6 ]. The end-replication problem is managed in most eukaryotes by the enzyme telomerase, which adds telomeric DNA sequences to the 3' end of chromosomes through the action of its catalytic subunit and RNA template (reviewed in [ 7 ]). DNA polymerase then forms duplex DNA by synthesizing the complementary C-rich strand of the telomere [ 8 ]. In fission yeast, the catalytic subunit of telomerase is encoded by the gene trt1 + [ 9 ]. In some cases, cells can endure the loss of telomerase and give rise to a population of survivors. In the budding yeast Saccharomyces cerevisiae , survivors maintain long, heterogeneous telomeres on linear chromosomes using a RAD52 -dependent homologous-recombination pathway [ 10 ]. Global gene-expression profiles of budding yeast lacking telomerase revealed the induction of a DNA damage response when telomeres were short and a sustained stress response in survivors [ 11 ]. Human alternative lengthening of telomeres (ALT) cells are cancerous cells lacking detectable telomerase activity that maintain long, heterogeneous telomeres using what is believed to be a strand invasion mechanism [ 12 , 13 ]. S. pombe cells without telomerase cease dividing after about 120 generations, and can give rise to a subpopulation of survivors [ 14 ]. Interestingly, these survivors have either circular chromosomes or linear chromosomes with long, heterogeneous amplified telomeres (presumably maintained through recombination) that resemble their budding yeast and human ALT-cell counterparts. While survivors with circular chromosomes arise more frequently, those with linear chromosomes grow faster [ 14 ]. Circular chromosomes in S. pombe are believed to form as a result of the genomic instability due to loss of telomeres, which normally prevent end-joining and suppress recombination. Interchromosomal fusions yield unstable dicentric chromosomes, while intrachromosomal fusions produce circular chromosomes. S. pombe , with only three chromosomes, is more likely than other organisms with larger numbers of chromosomes to successfully form exclusively intrachromosomal fusions [ 14 , 15 ]. S. pombe strains with circular chromosomes also result after concurrent deletion of rad3 + and tel1 + , two genes with sequence similarity to human ATM (ataxia telangiectasia mutated) [ 15 ]. Although S. pombe survivors with linear chromosomes grow remarkably well and have a morphology similar to wild-type cells, survivors with circular chromosomes display obvious growth defects such as slower growth rates and larger sizes [ 14 ]. Survivors with circular chromosomes presumably cope with impaired DNA segregation, and perhaps DNA breakage and rearrangement. We hypothesized that cells would show altered expression of genes necessary for coping with the loss of telomerase and concomitant changes in chromosome structure. In this study, we determined the S. pombe global gene-expression response to loss of trt1 + to investigate changes in expression of genes during senescence, and to compare survivors with circular or linear chromosomes. We report that survivors with circular chromosomes maintain an extended stress response not observed in survivors with linear chromosomes. Furthermore, we present evidence for regulation of a telomeric gene by the RNAi machinery. Results Wild-type reference strains Wild-type isogenic reference strains WT 3 and WT 5 were used to determine relative gene-expression changes in trt1 - samples. Before averaging the expression values from the two reference strains, the similarity of their expression profiles was assessed. The dye ratios measured by microarray for each strain were plotted against each other (Figure 1a ). All genes had expression values that varied less than twofold between the two samples, indicating that the samples were highly similar. The wild-type values used in this paper are thus the average expression values of strains WT 3 and WT 5. To learn whether changes in gene expression would result from subjecting cells to the continuous growth program for 15 days, gene-expression values from strain WT 5 on day 1 of the growth curve were compared with those of the same strain harvested on day 15 (Figure 1b ). Only three genes (SPBC354.08c, atp8 + and cox1 + ) changed their expression values by more than twofold, and they were only slightly greater; thus, the vast majority of genes do not have altered expression as a result of long-term growth in culture, provided that expression is measured while the cells are in early log phase (see Materials and methods). These three genes also had expression changes of more than twofold in one or more conditions measured for trt1 - cells, but given their variable expression in wild-type cells, these changes were most probably unrelated to the absence of telomerase. Watching cells pass through crisis and characterizing survivors Diploid S. pombe cells that were heterozygous for trt1 + and able to maintain full-length telomeres were sporulated, and the resulting trt1 + and trt1 - cells propagated through a 15-day growth curve (Figure 2a ). Cells lacking telomerase gave rise to survivors after day 8 concomitant with heterogeneous amplified telomeric repeats and telomere-associated sequence (TAS) (Figures 2b-d ), indicative of linear chromosomes [ 14 ]. By day 15, the culture was dominated by faster-growing cells with linear chromosomes. The linear structure of these chromosomes was confirmed by their ability to enter a pulsed-field gel (Figure 3b , lane g), and the existence of terminal chromosome fragments C, I, L and M after digestion of chromosomes with Not I (Figure 3a-d , lane e) [ 14 , 15 ]. Cells passing through crisis (days 7 and 9) also had weak hybridization signals for the C+M and I+L fragments (Figure 3d , lanes c-d), suggesting a mix of cells with either linear or circular chromosomes, or perhaps cells containing both linear and circular chromosomes. The inability to detect intact chromosomal DNA at day 7 (Figure 3b , lane e) may have resulted from the presence of cells with circularized chromosomes (Figure 3d , lane c) that do not enter pulsed-field gels. Strains C1 and C5 had circular chromosomes as evidenced by lack of telomeric repeats (data not shown), lack of TAS2 sequence (data not shown), the inability of chromosomes to enter a pulsed-field gel (Figure 3b , lanes b-c), the lack of terminal chromosome fragments C, I, L and M (Figures 3c,d , lanes g-h) [ 14 , 15 ], and hybridization signals to fragments C+M and I+L (Figure 3d , lanes g-h). Two waves of expression are observed in the growth curve Two waves of altered gene expression were seen during the growth curve (Figure 4a ), the first with a peak at day 7, consisting of around 110 genes with expression upregulated twofold or more, and the second with a peak at day 9, consisting of three microarray signals that appear to represent a single ORF (see below) (Figure 4a ). The peak of the first wave (day 7) was nearly coincident with crisis in the cell population (day 8) (Figure 2a ) and the time when telomeres were shortest (near day 7) (Figure 2c,d ). The second peak of gene expression at day 9 was coincident with the emergence of survivors (Figure 2a-d ). The vast majority of expression changes involved upregulation, and only seven genes had downregulated expression of twofold or greater on two or more days of the growth curve. Notably, there were three cases of reduction in expression greater than tenfold: trt1 + (intentionally knocked out), SPAC2E1P3.04 (a predicted copper amine oxidase) and SPAC2E1P3.05c (unknown function). Hybridizations of genomic DNA to microarrays (data not shown) revealed that genes SPAC2E1P3.04 and SPAC2E1P3.05c were deleted from the genome in all strains except WT 3, WT 5 and C1. Interestingly, these two genes are within about 4 kb of transposable element SPAC167.08 (Tf2-2), suggesting a hotspot for DNA excision. In no case was gene amplification detected by genomic hybridization (data not shown), so the observed increases in expression were most probably due to transcriptional or post-transcriptional regulation, as opposed to changes in gene copy number. Gene-expression changes in trt1 - cells Because a relatively large number of trt1 - strains were studied, the identification of genes with consistently altered expression was facilitated by selecting those genes with expression changes of twofold or more in two or more days of the growth curve or, alternatively, in both strains C1 and C5. This criterion was met by 123 genes, of which 54 (44%) overlapped between the growth curve and survivors with circularized chromosomes. In addition, of the 67 genes that had their expression changed twofold or more exclusively in the growth curve, many displayed altered expression just below the cutoff in survivors with circularized chromosomes. Two genes - SPBC1683.06c (a predicted uridine ribohydrolase) and SPBC1198.01 (a predicted formaldehyde dehydrogenase) - had expression changes of twofold or more in both strains C1 and C5, but no significant changes during the growth curve. As a measure of confidence, 84 of the 123 genes (approximately 68%) met a more stringent criterion requiring a gene to change its expression in three or more of the 17 conditions. Additional confidence that expression changes scored as significant were not false positives came from the remarkably continuous manner in which gene expression changed throughout the growth curve (Figure 4a ). The 123 genes with altered expression encompass a broad range of functions, but were especially enriched in genes associated with energy production and carbohydrate metabolism (Table 1 ). There were seven pseudogenes and 29 predicted genes that did not have assigned functions at the time of writing. For nearly all the gene-type categories, there was a larger number of genes with altered expression in the growth curve than in the survivors with circular chromosomes (Table 1 ). This difference may be attributable to the fact that cells in the growth curve were experiencing crisis whereas strains C1 and C5 were survivors, presumably with established mechanisms to cope with the absence of or the loss of telomeres. The telomerase-deletion response had a large overlap with genes that changed expression in response to environmental stresses. Fission yeast stress-response genes can be separated into a CESR, in which genes changed expression in all or most of the stresses studied (oxidative stress, heavy metals, heat shock, osmotic stress and DNA damage), and into more specific stress responses [ 16 ]. Of the 123 genes with altered expression in trt1 - cells, 48 (about 39%) also had upregulated expression among a conservative list of CESR genes ( P ~ 10 -77 ) [ 16 ], and two genes had downregulated expression in the CESR and in this study. Of the 110 genes with expression upregulated twofold or more on day 7 of the growth curve, 44% overlapped with the CESR. Comparison with a less conservative list of CESR genes [ 16 ] suggested that 54% of the 123 genes with altered expression in trt1 - cells had overlap with the CESR ( P ~ 10 -81 ). With respect to specific stress responses [ 16 ], there were 17/123 genes in common with the oxidative stress response ( P ~ 10 -32 ), and 11/123 genes in common with the heat stress response ( P ~ 10 -24 ). The stress response study found that the DNA damage response and the oxidative stress response have substantial overlap [ 16 ]. Therefore, the genes with altered expression in this study that overlap with the oxidative-stress response may represent a DNA damage response to short telomeres. Chromosome structure and gene expression Comparisons of all the gene-expression profiles in this study revealed striking differences between the profiles of survivors with linear chromosomes versus those with circular chromosomes. Survivors with linear chromosomes (days 12-15 of the growth curve) had gene-expression patterns similar to those of cells with native telomeres in the first two days of the growth curve. To illustrate, by day 12 of the growth curve, the gene-expression profiles of survivors became relatively constant and remained so through day 15. The profiles of days 12-15 appear most similar to days 1 and 2 of the growth curve, immediately after cells lost telomerase and were experiencing shortening telomeres (Figure 4b ). This observation was confirmed by hierarchical clustering (Figure 4c ). Conversely, survivors with circular chromosomes had gene-expression profiles that most resembled those of cells in crisis during days 5-8 of the growth curve (Figure 4b,c ). Sustained stress response in survivors with circular chromosomes There were 54 genes with clearly altered expression (twofold or more) mainly during crisis in the growth curve that also had altered expression in the survivors with circular chromosomes (Table 2 , Figure 5 ). The expression of all but three of these 54 genes was not altered in survivors with linear chromosomes (growth curve days 12-15) (Table 2 ). Of the 54 genes, 30 (56%) overlapped with the conservative list of CESR genes ( P ~ 10 -46 ), and eight genes (15%) overlapped with the oxidative stress response ( P ~ 10 -14 ). There were 8/54 genes (15%) that overlapped with the heat stress response ( P ~ 10 -17 ). Because of the extensive overlap of the 54 genes with the CESR, we conclude that survivors with circular chromosomes had a sustained stress response. Of the 54 genes, 51 represent a gene-expression signature that differentiates survivors with circular chromosomes from those with linear chromosomes. As an independent test of whether these 51 genes can serve as a signature for cells with circularized chromosomes, two additional cultures (strains H1 and H2, see Materials and methods) with circularized chromosomes were grown and analyzed by microarray. Both strains clearly displayed altered expression of the 51 genes whereas survivors with linear chromosomes did not (Figure 5 ), thus validating this gene signature. No altered expression of genes encoding recombination and telomere factors One feature of microarray studies is that genes not previously recognized to be under the control of a common regulator can often be associated by similar expression patterns [ 17 ]. On the basis of this hypothesis, a list of genes known to be involved in telomere maintenance and recombination was inspected. However, the expression patterns of all these genes were not substantially changed throughout the course of the study (data not shown). Genes investigated included pku70 + and lig4 + , which encode components of the non-homologous end-joining pathway [ 18 ]; taz1 + [ 19 ] and pot1 + [ 20 ] encoding telomere DNA-binding proteins; telomerase component est1 + [ 21 ]; homologous recombination-related genes rad22 + [ 22 ], rhp54 + [ 23 ], rad32 + [ 24 ] and rhp51 + [ 25 ]; RecQ helicase gene rqh1 + [ 26 ]; silencing component clr4 + [ 27 ]; and telomere maintenance components pof3 + [ 28 ] and rad3 + [ 15 ]. Interestingly, even though pof3 + and clr4 + expression did not change, the genes with altered expression in this study had a statistically significant overlap with the lists of genes with induced expression in pof3 mutants ( P < 10 -45 ) [ 28 ] and clr4 mutants ( P < 10 -45 ) [ 29 ]; a significant correlation was also observed with genes that changed expression in the RNA interference (RNAi)-machinery mutants dcr1 + , ago1 + and rdp1 + ( P ~ 10 -22 ) [ 29 ]. These genes with altered expression may act in common pathways downstream of trt1 + , clr4 + , pof3 + and the RNAi machinery. A second wave of expression represents sub-telomeric ORF with homology to RecQ helicases and dh repeats The second wave of gene-expression changes during the growth curve (Figure 4a ) consisted of three microarray signals: SPAC212.11 (largest magnitude), SPAC212.06 (second largest magnitude) and the reverse transcript of centromeric dh repeats [ 30 ]. Inspection of the sequences revealed that the microarray signals from SPAC212.06 and centromeric dh repeats most probably resulted from cross-hybridization with the SPAC212.11 transcript (see Materials and methods). A BLAST search of the SPAC212.11 predicted protein sequence found that the ORF has the most similarity to RecQ DNA helicases of superfamily II (Figure 6 ) (reviewed in [ 31 ]). We report a role for the helicase in cells passing through crisis in a separate study (J.G.M., K.J. Goodrich, J.B. and T.R.C., unpublished work) and investigate its transcriptional regulation here. SPAC212.11 is the last sequenced ORF on the left arm of chromosome I. The sub-telomeric regions of chromosomes I and II have significant similarity [ 32 ]. A BLAST search performed with the SPAC212.11 DNA sequence (5.6 kb) revealed a paralog, SPBCPT2R1.08c (6.3 kb), located on the right arm of chromosome II (the microarray had no probe for SPBCPT2R1.08c), and partial homology on the right arm of chromosome I. The annotated sequence of SPBCPT2R1.08c includes the entirety of the SPAC212.11 sequence with only a single base change. The SPAC212.11 sequence does not contain a stop codon because the ORF is located at the end of the sequencing contig, which ended before a stop codon was reached. Comparison with the annotated SPBCPT2R1.08c sequence suggests that SPAC212.11 has an additional 95 bp before the stop codon. Both SPBCPT2R1.08c and SPAC212.11 are the last predicted genes on their respective sub-telomeric sequencing contigs. Analysis of contig pT2R1 revealed that the 3' end of SPBCPT2R1.08c is approximately 2.8 kb upstream from the start of TAS3 (Figure 2b ). Since TAS3 is around 7 kb from the chromosome end, the 3' end of SPBCPT2R1.08c is approximately 10 kb from the telomeric repeats. It is not known which of the paralogs contributed to the SPAC212.11 microarray signal. For the sake of simplicity, further references in the text to 'the putative helicase' are meant to include SPAC212.11, SPBCPT2R1.08c and any paralogs, collectively. The nucleotide BLAST search performed with the SPAC212.11 sequence also revealed that the ORF contains regions of homology to dh repeats (Figure 6 ), which are targeted for heterochromatin formation via an RNAi-mediated mechanism in S. pombe [ 33 , 34 ]. These repeats are typically located at centromeres and the K region of the mating-type locus [ 30 , 33 , 35 - 37 ]. RNAi machinery implicated in controlling expression of the putative helicase Centromeric repeats, previously thought to be transcriptionally silent, are transcribed in both the forward and reverse directions, leading to formation of double-stranded RNA (dsRNA). However, these transcripts do not accumulate in wild-type cells. Reverse-strand centromeric transcripts are synthesized and rapidly processed by the RNAi machinery, while forward-strand synthesis is silenced transcriptionally. RNA-dependent RNA polymerase (Rdp1) associates with centromeric repeat DNA and may use siRNAs corresponding to centromeric transcripts [ 38 ] to prime forward transcription from reverse-strand templates, thus resulting in dsRNA formation and maintenance of the heterochromatic state. In the RNAi mutants dcr1 - , ago1 - and rdp1 - , centromeric silencing is abolished and accumulation of both forward and reverse centromeric transcripts is observed [ 33 ]. Microarray, northern blot and reverse transcription (RT)-PCR analysis indicated that the putative helicase gene was robustly expressed in cells emerging from crisis, but was weakly (or not at all) expressed in wild-type cells, strains C1 and C5 and survivors with linear chromosomes (Figures 4a,b , 7a , and data not shown). As the putative helicase transcript was not detectable by northern blot in wild-type cells (data not shown), we hypothesized that this ORF could be silenced by its dh repeats, but that this silencing may have been disrupted in trt1 - cells as a result of genomic instability. Arguing against this hypothesis, however, Southern analysis with probe P 5' (Figure 6 ), which is specific for the helicase, did not reveal any DNA rearrangements during crisis close to the helicase that might have contributed to loss of silencing (data not shown). Nevertheless, the loss of silencing observed might lead to expression of both strands of the putative helicase, as was found for centromeric dh repeats in RNAi mutants. To test for the presence of both strands, strand-specific RT-PCR was used with primers spanning the dh repeats of the putative helicase (region P dh in Figure 6 ). The forward strand was expressed at levels higher than in wild type in cells from days 7, 9 and 15 of the growth curve. These results were consistent with microarray analysis that detected the 3' end of the forward transcript (Figure 7a ). The reverse strand was weakly detectable in cells from days 7 and 9 of the growth curve (Figure 7a ). dsRNA arising from the repeats presumably could have formed on days 7 and 9 of the growth curve, but why such RNA was not all processed by the RNAi machinery is not clear. On days 7 and 9 of the growth curve, the RNAi machinery was not apparently affected by the mutation of telomerase as centromeric dh repeat transcripts were not detected by RT-PCR (Figure 7a ). We next hypothesized that if the RNAi machinery were involved in transcriptional silencing of the putative helicase in wild-type cells, transcript should accumulate in mutant RNAi strains. Strikingly, both ago1 - and dcr1 - strains displayed significant accumulation of the forward transcript of the putative helicase, and the rdp1 - strain showed slightly increased accumulation with respect to wild-type (Figure 7b ). The reverse strand did not accumulate in these three strains. Thus, transcriptional silencing of the putative helicase appeared to be relieved in RNAi mutants, implicating RNAi in the control of expression of this ORF. Discussion Correlation of chromosome structure and gene expression The genome-wide survey of expressed genes in this study provided an opportunity to investigate the cellular response to loss of the gene for the telomerase catalytic subunit Trt1. A major finding was the tight correlation between the structures of chromosomes in survivors and gene expression profiles. Survivors with linear chromosomes had expression profiles remarkably similar to cells with canonical - yet shortened - telomeres, whereas cells with circular chromosomes maintained the upregulated expression of a significant number of genes that also had upregulated expression during senescence. The stress response in survivors with circular chromosomes had significant overlaps with the S. pombe CESR and with the heat and oxidative stress responses. The CESR consists of genes that had upregulated expression in all or most responses to oxidative stress, heavy metal stress, heat shock, osmotic stress and DNA damage [ 16 ]. The stress response may persist in survivors with circularized chromosomes because of impaired DNA segregation and DNA breakage and rearrangement. Indeed, compared with wild-type cells, survivors with circular chromosomes are larger and have slower growth rates, indicating that functions related to cell division are impaired [ 14 ]. Telomeric repeats contribute to recruiting the molecular components collectively involved in the protective capping of chromosome ends [ 20 , 39 , 40 ]. These repeats are maintained in the absence of telomerase in cells from diverse organisms that normally use telomerase (reviewed in [ 3 ]). Interestingly, the survivors with linear chromosomes abated their stress response concomitant with the appearance of amplified telomeric and TAS repeats as rare survivors took over the population, suggesting that the repeats helped to ameliorate the stress response. Neither cells in the growth curve that experienced shortened telomeres nor survivors with long telomeres displayed upregulation of telomeric gene expression, supporting the notion that telomeric length changes alone do not affect gene expression in S. pombe [ 19 ]. In addition, in survivors with circular chromosomes, only eight microarray signals, corresponding to as few as two genes (due to cross-hybridization) near former telomeres had altered expression, although such changes might have been expected as a result of the large alterations in chromosome structure at these sites. Comparison with the budding yeast response to loss of telomerase As in fission yeast, genes with changed expression in the budding yeast response to loss of telomerase had significant overlaps with genes whose expression was altered by environmental stresses such as heat shock, osmotic shock, dithiothreitol (DTT) , nitrogen starvation and peroxide ([ 11 , 41 ] see also [ 42 ]). A difference in the stress responses between the two yeasts was that in budding yeast a large but specific subset of the environmental stress-response genes persisted in survivors with linear chromosomes four days after crisis, whereas in fission yeast survivors with linear chromosomes, the stress response mostly abated by the fourth day after crisis (Figure 4b , day 12). The different yeast responses may be due to a fission yeast telomere structure that was not as strongly recognized as aberrant, perhaps mitigating a DNA-damage response. It is also possible that had budding yeast survivors been followed longer, providing a period for adaptation, the stress response would have subsided. In fission yeast, the expression of a number of mitochondrial ATP synthase genes was upregulated (Table 1 ) with orthologs similarly induced in budding yeast. In both cases, the changes did not overlap with the DNA-damage responses of the yeasts, further supporting a link between short telomeres and alterations in the metabolic program suggested by Nautiyal et al . [ 11 ]. Significance of putative RecQ helicase RecQ helicases have recently been implicated in telomerase-independent telomere maintenance in both S. cerevisiae and human ALT cells. BLM and WRN, human RecQ helicases associated with cancer and disease [ 31 ], have both been shown to associate with duplex telomere repeat binding protein TRF2 in vivo , and BLM co-localizes to telomeric foci exclusively in ALT cells [ 43 - 45 ]. The S. cerevisiae ortholog of human WRN and BLM, Sgs1, was also shown to be required for telomere elongation of type II survivors in the absence of telomerase [ 46 - 48 ]. The long, heterogeneous telomeres of S. pombe survivors with linear chromosomes are similar to those of S. cerevisiae survivors and human ALT cells, suggesting a role for RecQ helicases in fission yeast telomerase-independent telomere maintenance. dh repeats and RNAi at the telomere This is the first report to our knowledge of naturally occurring dh repeats outside of the centromeric and mating-type regions in fission yeast. We have presented several results that suggest that sub-telomeric dh repeats promote heterochromatin formation at the helicase locus. First, transcript from this ORF was only weakly expressed in wild-type cells as determined by RT-PCR (Figure 7a ) (and was not detectable at all by northern hybridization, data not shown), consistent with transcriptional regulation of this ORF by heterochromatin. Second, expression of the putative helicase was robust in ago1 - and dcr1 - mutants, which would be expected if RNAi has a role in transcriptionally silencing this ORF. In trt1 - mutants experiencing genomic instability, we detected both forward and reverse transcripts of sub-telomeric dh repeats (Figure 7a ). The presence of these complementary transcripts suggests the existence of dsRNA that had not been processed by the RNAi machinery, consistent with a lack of silencing at this locus. Intriguingly, after maximal expression of both strands on day 9 of the growth curve, subsequent downregulation was observed by day 15 (Figure 7a ), consistent with restoration of silencing. While the finding of homology with dh repeats at the sub-telomere was unexpected, dh repeats have been shown to function in silencing at sites outside of centromeres and the mating-type locus. Reporter genes fused to centromeric repeat fragments as short as 580 bp were silenced when integrated at ectopic locations in the genome [ 49 , 50 ] and this silencing required the RNAi machinery [ 51 , 52 ]. The longest (nearly continuous) stretch of sequence with homology to dh repeats found in the helicase ORF was about 600 bp (Figure 6 ), presumably long enough to promote heterochromatin formation. In addition, RNAi-mediated silencing triggered by both a synthetic hairpin RNA and transposon long terminal repeats have been shown to induce heterochromatin formation away from centromeres and the mating-type locus [ 53 ]. In a separate study, telomeric silencing of a reporter gene and binding of Swi6 at the telomere were not affected in dcr1 - , ago1 - and rdp1 - mutants [ 54 ]. The lack of an observed effect may have been due to the ability of telomeric repeats to recruit silencing factors. Indeed, telomeric heterochromatin is largely promoted by telomeric repeats. However, the study by Hall and co-workers [ 54 ] did report defective mitotic and meiotic telomere clustering in RNAi mutants, supporting a role for RNAi at telomeres. Given the correlation between disruption of telomeric heterochromatin and expression of the helicase ORF, events other than telomere erosion that disrupt heterochromatin might also induce helicase expression. Materials and methods Strain construction The trt1 + and trt1 - cells used in this study were generated by sporulating S. pombe diploid strain G4 ( h - / h + ade6-M210 / ade6-M216 trt1 + / trt1 - ) on ME plates [ 18 ]. The parent diploid strain was made heterozygous for trt1 + by using a standard two-step integration procedure [ 55 ] with a linearized plasmid containing about 1 kb each of the 5' and 3' flanking regions of the trt1 + ORF separated by HSV1-tk and KanMX4 [ 56 ]. The plasmid was linearized in the middle of the 3' flanking region with Fse I and transformed using the lithium acetate method [ 57 ] into a diploid strain created by crossing PP68 ( h - ade6-M210 ) and PP69 ( h + ade6-M216 ). Cells were re-streaked twice on yeast extract low adenine (YEA) + geneticin plates [ 18 ] to select for stable genomic integrants, which were subsequently confirmed by Southern hybridization to a uniquely sized Eco RI restriction fragment 3' of trt1 + which was present only in integrants. Cells were then plated on YEA + 50 μM 5-fluorodeoxyuridine (5-FUdR) plates to select for those that had excised HSV1-tk , KanMX4 and the Xba I- Xho I fragment (around 5 kb) of trt1 + from their genomes. Random surviving colonies were screened for heterozygous diploids by Southern hybridization to the 3' region of the trt1 + Kpn I restriction fragment. The heterozygous state was evidenced by hybridization signals to both full-length trt1 + and a shortened, nonfunctional version. Loss of markers was confirmed by lack of a Southern hybridization signal to HSV1-tk , and by lack of growth on YEA + geneticin plates. Selection of strains After germination of G4 and growth of spores at 32°C for three days on YEA plates [ 18 ], plates were stored at 4°C while the genotypes of random colonies were determined. A portion of single colonies was used for crossing and visual inspection to identify those that had an h - ade6-M210 genotype, which were further screened by Southern hybridization for the presence or absence of trt1 + (performed as described in 'Strain construction' below). Colonies were subsequently used as described in 'Growth curve', or alternatively used to create strains C1, C5, H1 and H2. Strains C1, C5, H1 and H2 were created from four separate trt1 - colonies that were each successively re-streaked on YEA plates 15 times (with growth for 2 to 3 days at 32°C between re-streaks), to permit colonies to form without competition from faster-growing survivors with linear chromosomes. During this time cells were presumed to senesce and give rise to survivors. After the last re-streak, a single colony from each strain was randomly selected and used to prepare freeze stocks. Growth curve Three strains were grown: two wild-type isolates ( h - ade6-M210 trt1 + ) designated WT 3 and WT 5, and a single mutant isolate ( h - ade6-M210 trt1 - ) designated as 'GC Day X', where X represents the day of the growth curve that cells were collected. Single colonies were used to inoculate 5-ml starter cultures in yeast extract full supplements (YES) medium [ 18 ] and grown for 24 h with shaking at 32°C. Cells were counted and used to inoculate 200-ml YES cultures in 500-ml Erlenmeyer flasks at 2.5 × 10 4 cells/ml, and were grown in an incubator (Innova 4430, New Brunswick Scientific) with continuous shaking at 200 rpm at 32°C. Cell density was monitored by periodic counting, and a portion of the cells was harvested for microarray analysis and Southern hybridization when the density reached 3-5 × 10 6 cells/ml (early log phase). Cells harvested at this point were referred to as day 1 of the growth curve. The unharvested cells were permitted to continue growing until 24 h from the time of inoculation, at which time cells were counted and used to inoculate a fresh 200-ml YES culture at 2.5 × 10 4 cells/ml, and the process repeated for 15 days. To harvest cells for microarray analysis, a volume of culture containing approximately 1.6 × 10 8 cells was gently centrifuged at room temperature (2,000 rpm for 2 min), the supernatant removed, and the cell pellet snap-frozen in liquid N 2 . For Southern hybridization, approximately 2 × 10 8 cells were collected by centrifugation, washed twice in H 2 O, and snap-frozen in liquid N 2 . A portion of cells for pulsed-field gel analysis was also collected in the same manner as for Southern hybridization at the end of each 24 h period. trt1 - cells were collected daily, WT 3 and WT 5 on day 1, and WT 5 on day 15. Growth and collection of strains C1, C5, H1 and H2 Cells were streaked onto YEA plates from freeze stocks, grown for 3 days at 32°C, and single colonies used to inoculate 5-ml starter cultures in YES medium. After 24 h, cells were counted and 200-ml YES cultures were inoculated at 2.5 × 10 4 cells/ml, and cultures grown with constant shaking at 200 rpm at 32°C. When the cell density reached around 3 × 10 6 cells/ml (early log phase), cells were collected as described in 'Growth curve' for microarray analysis, Southern hybridization and pulsed-field gel electrophoresis. Strains H1 and H2 are trt1 - isolates with circular chromosomes, as evidenced by pulsed-field gel electrophoresis (data not shown) Genomic DNA preparation and Southern hybridization DNA from approximately 2 × 10 8 S. pombe cells was prepared as described [ 18 ]. After digestion with either Eco RI or Hind III, the DNA was subjected to electrophoresis on a 1% agarose gel in 1 × TBE (90 mM Tris, 90 mM borate, 2 mM EDTA pH 8.3). DNA was denatured by sodium hydroxide treatment and transferred to a nylon membrane (Hybond-N+ membrane, Amersham) by capillary transfer in 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate). DNA was immobilized on the membrane by irradiation with 120 mJ/cm 2 at 254 nm in a UVStratalinker1800 (Stratagene). For molecular weight markers, the 1 kb DNA ladder (New England Biolabs) was labeled by filling in 5' overhangs with [α- 32 P]dATP using DNA polymerase I Klenow fragment. Probes for pol1 + , act1 + , the putative helicase and the C, I, L and M chromosome fragments were generated by PCR amplification from a genomic DNA template and were gel purified. Probes were labeled by random-primed transcription of PCR products with the use of [α- 32 P]dCTP and High Prime Mix (Boehringer Mannheim). Probes specific for the telomeric and telomere-associated sequences were created with the use of gel-purified fragments of pNSU70 [ 58 ]. Pulsed-field gel electrophoresis Cells (approximately 1 × 10 8 ) were collected as described above. Plug preparation, chromosome digestion and electrophoresis were performed exactly as described [ 18 ]. DNA was visualized by staining with ethidium bromide (1 μg/ml) for 30 min. The gel was then irradiated with 120 mJ/cm 2 at 254 nm in a UVStratalinker1800 to nick the DNA, treated with HCl, NaOH and neutralization buffer, and processed as described in 'Southern hybridization'. RT-PCR RNA was prepared as for microarray analysis and used for RT-PCR (OneStep RT-PCR kit, Qiagen). First-strand cDNA synthesis was performed using primers complementary to either the forward or reverse strands. Both primers were present in subsequent cycles of PCR amplification after heat inactivation of reverse transcriptase at 95°C for 15 min. The control reaction lacking reverse transcriptase ( act1 + sense, -RT) was not subjected to first-strand cDNA synthesis, but was otherwise treated identically. Probes and PCR primers The PCR primers used to generate probes C, I, L, and M have been published previously [ 14 ]. The PCR primers spanning the regions described in Figure 6 were: P 5' : 5'-CTTCAAAAACTGCTAGAGATATCGCCGG-3' and 5'-GTACTGGTAGTCCTCTGATGTATGGG-3' P 3' : 5'-ATGCCCCGTACGCTTATCTA-3' and 5'-TTTGCCTTTCTAGCCCATGA-3' P dh : 5'-CAACACCAATACTGACGATGATG-3' and 5'-GCAATAGAACCAGCGGTTTG-3' Primers for centromeric dh repeats have been published previously [ 33 ]. RNA preparation and reference pool for microarrays Whole-cell RNA was isolated from S. pombe cell pellets (~1.6 × 10 8 cells) by hot-phenol extraction and purification with RNeasy columns (Qiagen) following a published protocol [ 59 ]. Aliquots (10 μg) were made (henceforth referred to as 'sample RNA') and RNA quality was assessed by UV absorbance, by agarose gel electrophoresis to confirm intact rRNA bands, and by northern hybridization to act1 + . A reference pool consisting of RNA from each sample was made, comprising 76% trt1 - cells and 24% trt1 + cells. This pool was divided into 10 μg aliquots (henceforth referred to as 'reference RNA') and used as the reference RNA in all hybridization experiments reported here. A single large batch of YES medium was made at the start of the study and used to culture all cells analyzed by microarrays to prevent batch-to-batch medium variations that might yield artifactual microarray results. Microarray cDNA labeling, hybridization and data acquisition The procedures performed and the S. pombe microarrays used have been described previously [ 59 ]. Whole-cell RNA (10 μg) was labeled by directly incorporating either Cy3-dCTP (reference RNA) or Cy5-dCTP (sample RNA) through reverse transcription. The resulting cDNA was hybridized onto DNA microarrays containing spotted PCR products for over 5,269 different genes and genomic elements printed in duplicate on glass slides representing 99.9% of all known and predicted fission yeast genes. Microarrays were scanned using a GenePix 4000B laser scanner (Axon Instruments) and analyzed with GenePix Pro software. Low-quality signals were filtered out, and data were normalized using a customized Perl script (local adjustment of median of ratios to one within running windows of 1,000 spots). Data evaluation and gene classification Normalized data (Cy5/Cy3 ratios) were evaluated using GeneSpring (Silicon Genetics). All gene-expression values were normalized to the average of two trt1 + biological replicates (strains WT 3 and WT 5) collected on day 1 of the growth curve. Experiments and genes were clustered in GeneSpring using the Pearson correlation around zero (termed the Standard correlation in GeneSpring) with a minimum distance of 0.001 and a separation ratio of 1. Gene annotations were taken from GeneDB at the Wellcome Trust Sanger Institute [ 60 ]. Lists of genes whose expression changed in the fission yeast stress response [ 16 ] were taken from the authors' website [ 61 ]. BLAST searches were performed using the NCBI BLAST server [ 62 ]. The density of genes with changed regulation along the chromosome was determined by using a running window of 20 consecutive genes along each chromosome [ 63 ]. For each window, the probability of obtaining the observed results by chance was calculated using the hypergeometric distribution. There were two microarray signals - SPAC212.06 (a pseudogene) and the reverse transcript of centromeric dh repeats - that we believe were due to cross-hybridization with the SPAC212.11 transcript (or transcripts from identical ORFs, see text). Cross-hybridization becomes apparent with array element sequence identities higher than about 70% [ 59 ]. The SPAC212.11 transcript is capable of hybridizing to the entire SPAC212.06 microarray probe (99% sequence identity), but the SPAC212.06 transcript does not contain the sequence required to hybridize to the SPAC212.11 microarray probe. The SPAC212.11 transcript also has a high degree of homology with the dh repeat microarray probe sequence (83% sequence identity), and both the SPAC212.11 and dh repeat transcripts are expected to be capable of hybridizing to each other's microarray probes. Significant levels of forward and reverse centromeric dh repeat transcripts could not be detected using RT-PCR with RNA from days 1, 7, 9 and 15 of the growth curve (Figure 7a ) (indicating they could not hybridize to the microarray), although helicase RNA was detected by both RT-PCR and northern hybridization (Figure 7a and data not shown). Twenty-one microarrays were used in this study, representing two wild-type biological repeats, 15 days of the growth curve, and four strains with circularized chromosomes. The complete raw and normalized data sets are available from ArrayExpress [ 64 ] (Accession number: E-MEXP-201). | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549061.xml |
535343 | Efficacy of repeated intrathecal triamcinolone acetonide application in progressive multiple sclerosis patients with spinal symptoms | Background There are controversial results on the efficacy of the abandoned, intrathecal predominant methylprednisolone application in multiple sclerosis (MS) in contrast to the proven effectiveness in intractable postherpetic neuralgia. Methods We performed an analysis of the efficacy of the application of 40 mg of the sustained release steroid triamcinolone acetonide (TCA). We intrathecally injected in sterile saline dissolved TCA six times within three weeks on a regular basis every third day in 161 hospitalized primary and predominant secondary progressive MS patients with spinal symptoms. The MS patients did not experience an acute onset of exacerbation or recent distinct increased progression of symptoms. We simultaneously scored the MS patients with the EDSS and the Barthel index, estimated the walking distance and measured somatosensory evoked potentials. Additionally the MS patients received a standardized rehabilitation treatment. Results EDSS score and Barthel index improved, walking distance increased, latencies of somatosensory evoked potentials of the median and tibial nerves shortened in all MS patients with serial evaluation (p < 0.0001 for all variables). Side effects were rare, five patients stopped TCA application due to onset of a post lumbar puncture syndrome. Conclusions Repeated intrathecal TCA application improves spinal symptoms, walking distance and SSEP latencies in progressive MS patients in this uncontrolled study. Future trials should evaluate the long-term benefit of this invasive treatment. | Background There are controversial results on the efficacy of the nowadays still abandoned, intrathecal steroid application predominantly due to a missing detailed evaluation of patients' characteristics, careful monitoring and standardized outcome measurements in multiple sclerosis (MS) [ 1 ]. Initially, case reports of intrathecal methylprednisolon and ACTH administration described beneficial effects in MS patients, but the following studies showed disappointing results in particular in comparison to systemic steroid application [ 1 ]. Earlier intrathecal treatment trials in MS patients suffered from small sample sizes, low number of injections, low steroid dosages, short half life of the administered steroid and an increasing number of reports on side-effects, i.e. adhesive arachnoiditis and various forms of meninigitis probably due to neurotoxic solvents and bacteriostatic additives [ 4 ]. Moreover a retarded release steroid preparation was not available for many years [ 1 , 5 ]. Then administration of triamcinolone acetonide crystal suspensions (TCA), dissolved at bedside in sterile saline, was introduced in the intrathecal steroid treatment of MS. However, studies again lacked of detailed evaluation and clinical characterization of MS patients, small sample sizes and a low number of intrathecal application of this retarded release steroid compound [ 1 , 4 , 5 ]. Accordingly, there was no convincing superiority over the efficacy of the systemic steroid treatment. Some enrolled MS patients experienced a recent deterioration of symptoms due to prior acute relapses and/or ongoing chronic progression. Moreover study participants were not classified according to the various subtypes of MS progression [ 5 , 6 ]. However in general, a comparison of both methods of steroid application is at least doubtful from the pharmacokinetic point of view. The resulting steroid efficacy in the central nervous system enormously differs in favor for the intraspinal application due to the achieved cerebrospinal fluid steroid level, marked longer half life, i.e. with detection of TCA even four months after the last administration, and missing impact on the endogenous peripheral cortisol secretion with no appearance of side effects of systemic high dosage steroid application [ 4 , 6 ]. In recent years, a certain revival of intrathecal methylprednisolon administration took place in the treatment of intractable postherpetic neuralgia and MS with spinal symptoms, both of which turned out to be very effective, but were controversially discussed regarding the safety issues [ 2 , 3 ]. The MS study demonstrated, that six repeat intrathecal TCA injections within three weeks reduced the EDSS score in 31 of 36 progressive MS patients with predominant spinal symptoms. 20 of them entered a follow-up period of 13.1 ± 6.22, 3 – 23 [mean ± SD, range] months with 6.35 ± 3.91, 2 – 15 TCA injections. They received one TCA application on a regular basis in an individually differing frequency every six to twelve weeks. These patients remained stable [ 3 ]. Nevertheless, there is a need for further results on the usefulness of this treatment. The optimum design would be a placebo-controlled arm, but repeat performance of intrathecal saline (placebo) administration under double-blind conditions with the patients' consent and an approval of an ethical committee is not realistic in clinical practice. Moreover they may be ethical concerns of withholding treatment [ 7 ]. Our present study is a way out of this dilemma of the debate on the efficacy of TCA treatment, which is carried out in certain specific centers in Germany for many years now [ 3 ]. We performed an analysis of standardized intrathecal application of TCA in MS patients with spinal symptoms, using subjective rating procedures and objective measurements. Methods Subjects We only enrolled clinically well characterized, consecutively referred MS patients (table 1 , 2 ) with distinct spinal symptoms and/or MRT visualized lesions in the spinal cord [ 8 ]. These patients did not suffer from an acute onset of exacerbation or recent clearly increased progression of their symptoms. Table 1 Patients' characteristics. Age 50.10 ± 10.30, 21 – 78 years Duration of MS 14.32 ± 7.63, 2 – 40 years Sex 119 men, 42 women MS types chronic progressive: n = 35 secondary progressive: n = 122 relapsing-remitting: n = 4 Length of hospital stay 28.41 ± 5.97; 21 – 60 days Table 2 Treatment against spasticity. Before TCA After TCA Baclofen 7.56 ± 16.06; 0 – 80 mg, n = 117 without baclofen 6.44 ± 15.51; 0 – 80 mg, n = 125 without baclofen Tolperison 1.19 ± 8.20; 0 – 75 mg, n = 154 without tolperison 1.34 ± 7.41; 0 – 50 mg, n = 152 without tolperison Tizanidin 1.94 ± 5.29; 0 – 32 mg, n = 132 without tizanidin 3.38 ± 6.64; 0 – 32 mg, n = 110 without tizanidin All data are given as mean ± standard deviation; minimum – maximum; n = number of patients; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section. Methods We performed scoring with both, EDSS and Barthel index and assessed the walking distance. Then we measured somatosensory evoked potentials (SSEP) in a standardized fashion before start and at the end of the intraspinal TCA treatment within a prospective study design [ 9 ]. A technician performed SSEP recordings and measured the walking distance. We blinded the EDSS raters. Retrospectively, we compiled information on patients from their hospital records, i.e. date of birth, sex, duration of disease after diagnosis of MS, dosages of oral baclofen (lioresal ® ), tolperison (mydocalm ® ), tizanidin (sirdalud ® ) on the first and last day of the hospital stay, length of hospitalization in days (tables 1 & 2 ). The patients additionally received a standardized rehabilitation treatment, which included physiotherapy, massage and optional swimming with the patients' consent [ 12 , 13 ]. Only data with successfully performed serial evaluation of patients were compared for each variable. We performed lumbar puncture with an "atraumatic" Sprotte needle [ 10 , 11 ]. Each patient received six intrathecal applications of 40 mg TCA followed by a mandatory stay in bed for at least six hours. This should reduce incidence of lumbar puncture syndrome and hypothetically support the diffusion of TCA in the CSF and the spinal cord [ 14 , 15 ]. A preexisting immune system modulating drug therapy remained stable. We closely monitored for typical concomitant with systemic steroid application appearing side effects, i.e. increase of body weight etc., all of which did not significantly change (data not shown) [ 16 ]. No slight or severe side effects occurred, but we did not include five patients into our evaluation due to onset of post lumbar puncture syndrome with headache and nausea, which caused a stop of further intrathecal TCA applications. These patients withdraw their consent. We only considered SSEP data of patients with serial measurements, which we performed on the same day of the EDSS rating. Ethics Each participant gave written informed consent for the TCA treatment, which was approved by the local ethical committee. The consent form included a detailed description of all putative risks of lumbar puncture and intrathecal TCA application. Statistics Data showed a normal distribution according to the Kolmogorow-Smirnow test. As a result, we only performed parametric tests. We used ANCOVA with repeated measures design including MS duration, MS types, change of dosages of concomitant drugs against spasticity, length of hospital stay, sex and age as covariates. We computed SSEP results by adding both sides in order to reduce amount of calculations for comparisons. Then we calculated the differences of latencies between both timepoints of recordings according to the formula [Initial - End = Diff] for correlation analysis. We employed linear regression for correlation analysis. Level of significance of p-values were adjusted to 0.05 divided by the number of performed comparisons respectively correlations. Results Comparisons EDSS score (n [number of subjects with serial evaluation] = 161) and Barthel index (n = 68) improved and walking distance (n = 161) increased (table 3 ). SSEP latencies of tibial (n = 136) and median (n = 108) nerves reduced (table 3 ). P-values of all performed comparisons were below 0.0001. No significant effects of covariates appeared. Table 3 Comparison of clinical data. Before TCA After TCA F EDSS – score 6.44 ± 1.06; 3.5 – 6.5 5.47 ± 1.24; 2 – 8.5 379.28 walking distance 158.03 ± 501.20; 0 – 5000 439.38 ± 895.24; 0 – 5000 34.40 Barthel index 58 ± 20.07; 5 – 100 89.13 ± 12.57; 60 – 100 347.52 P2 (tibial nerve) 105.80 ± 10.38; 85 – 130 88.57 ± 5.60; 77 – 106 470.96 N2 (tibial nerve) 118.83 ± 10.49; 92 – 148 101.63 ± 6.43; 80 – 118 425.71 P3 (tibial nerve) 131.79 ± 13.22; 70 – 164 114.31 ± 7.46; 95 – 135 325.88 N2 (median nerve) 46.53 ± 5.40; 23–60 41.03 ± 2.92; 23–47 138.10 P2 (median nerve) 52.73 ± 6.34; 25 – 68 47.09 ± 3.03; 41–56 128.80 All data are given as mean ± standard deviation; minimum – maximum in the second and third column; latencies (N2, P2, P3) of the somatosensensory evoked potentials are given in milliseconds, walking distance is given in meters, F = F-value of ANCOVA, SD = standard deviation; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section. Correlation analysis There were no significant relations between computed changes of EDSS scores, walking distances and SSEP results (results not shown). Diff P2 correlated with Diff P3 (R [correlation coefficient] = 0.71), Diff P2 with Diff N2 (R = 0.90), Diff P3 with Diff N2 (R = 0.74) of SSEP latencies of the tibial nerves. There were relations between Diff N2 and Diff P2 (R = 0.85) of SSEP latencies of the median nerves. P-values of these correlations were below 0.0001. Moreover Diff P2 of the tibial nerves correlated with Diff N2 of the median nerves (R = 0.28, p = 0.004, n = 100). There was a certain trend for a significant correlation between Diff P2 of the tibial nerve and Diff P2 of the median nerve (R = 0.22, p = 0.03). No other significant associations of SSEP data appeared (results not shown). Discussion Our results demonstrate and confirm the efficacy of repeated intraspinal TCA application in MS patients with spinal symptoms, which improved according to the EDSS outcomes and the results of assessed walking distance and determined SSEP latencies [ 3 ]. We intrathecally injected TCA six times within three weeks, whereas earlier trials weekly performed one application up to three times at the most [ 4 ]. The distinct reduction of SSEP peak latencies and the significant relations between their computed differences confirm the clinical outcomes and underline the efficacy of intraspinal TCA treatment in MS patients with spinal symptoms in general. We hypothesize, that these neurophysiological results indicate a certain remyelinating and/or restorative potential of intraspinal TCA application with an at least transient shift from chronic inflammation to remyelination [ 17 , 18 ]. Our results support the crucially discussed view, that serial SSEP studies in MS may monitor the effect of treatment to a certain extent under standardized conditions [ 9 , 19 , 20 ]. Our analysis also shows that primary and secondary progressive even advanced MS patients with spinal symptoms predominantly improve from this kind of intrathecal steroid therapy. We assume, that we achieve persistent high steroid concentrations at lesions of the spinal cord, since TCA must not pass the blood brain barrier [ 6 ]. However previous comparisons of the clinical efficacy of intrathecal TCA application with the intravenous administration of methylprednisolone showed no superiority of one method over the other [ 5 , 6 , 19 ]. But these studies did not exclude relapsing remitting patients or participants with a previous acute relapse. They did not focus on spinal symptoms. Their application rate of TCA was distinct lower compared with the one of our present and a previous trial [ 3 ]. However our present study outcomes do not allow any conclusions on the duration of the achieved benefit and the impact of TCA treatment on progression of MS [ 3 ]. Therefore there is an urgent need for further confirmatory trials, which additionally address all these issues. A strategy would be to choose one arm with active treatment and one arm with just follow-up without active treatment with blind assessment by an evaluating physician. However we stress concerning long-term steroid therapy and progression of MS, that there are positive outcomes of trials with intravenous methylprednisolone administration in various application rates and dosages on long term disease progression and/or on brain atrophy in secondary-progressive -, respectively relapsing-remitting MS patients [ 16 , 21 ]. In contrast to studies on intravenous oral steroid treatment, we did not observe the typical side effects of systemic high dosage steroid administration, i.e. edema. This may support previous findings by circumstantial evidence, which report no decrease of endogenous cortisol secretion following intrathecal TCA administration [ 4 ]. We cannot exclude a certain impact of physiotherapy, the standardized rehabilitation treatment and an beneficial effect of hospitalization in general with its resulting concomitant positive influence on activities of daily living [ 12 , 13 ]. However, we found no significant impact of the corresponding covariate length of the hospital stay in our statistical analysis. We assume, that our results do not reflect an improved drug therapy against spasticity, since no significant impact of the covariate computed changes of medication appeared. Nevertheless we cannot exclude a certain effect of the steroid on spasticity. However most participants did not take any drug against spasticity. Onset of side effects of lumbar puncture itself were negligible, since we used an atraumatic needle [ 11 ]. Conclusions Our data demonstrate the efficacy and safety of repeated intrathecal TCA application in MS patients with predominant spinal symptoms, which markedly improved. Some MS patients experienced post lumbar puncture syndrome with a frequency within the normal range [ 11 ], but typical side effects of systemic high dosage steroid administration did not appear. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KH, FS, HP and TM designed, coordinated and carried out the study. TM performed statistical data analysis and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535343.xml |
549049 | Revisiting immunosurveillance and immunostimulation: Implications for cancer immunotherapy | Experimental and clinical experience demonstrates that the resolution of a pathogenic challenge depends not only on the presence or absence of an immune reaction, but also on the initiation of the proper type of immune reaction. The initiation of a non-protective type of immune reaction will not only result in a lack of protection, but may also exacerbate the underlying condition. For example, in cancer, constituents of the immune system have been shown to augment tumor proliferation, angiogenesis, and metastases. This review discusses the duality of the role of the immune system in cancer, from the theories of immunosurveillance and immunostimulation to current studies, which illustrate that the immune system has both a protective role and a tumor-promoting role in neoplasia. The potential of using chemotherapy to inhibit a tumor-promoting immune reaction is also discussed. | If only it were all so simple. If only there were evil people somewhere insidiously committing evil deeds, and it were necessary only to separate them from the rest of us and destroy them. But the line dividing good and evil cuts through the heart of every human being, and who is willing to destroy his own heart? Alexander Solzhenitsyn, The Gulag Archipelago The notion that the immune system may be manipulated into recognizing and eradicating neoplasia is not new. Heroic efforts to develop a cancer vaccine can be traced as far back as 1777 when the surgeon to the Duke of Kent injected himself with malignant tissue as a prophylaxis against development of cancer. In 1808, another attempt was made to develop a cancer vaccine by the doctor to Louis XVII who inoculated himself with breast cancer in hope of reversing a soft-tissue sarcoma, although no therapeutic effect was observed. However, it was not until 1891 that the first report of successful immunotherapy was published by William Coley, a clinician at the Memorial Sloan Kettering Cancer Institute in New York. Using heat-killed endotoxin-containing bacteria (streptococci and Serratia marcescens ), Coley was able to achieve a cure rate of 10% in soft-tissue sarcoma [ 1 , 2 ]. Nevertheless, despite the numerous attempts over the past centuries to use the immune system in the eradication of cancer, the success rate of cellular immunotherapy remains abysmally low. In light of the successes in the development of vaccines targeting pathogenic agents, this review suggests that lessons learned from the immunology of infectious disease may be applicable to the treatment of neoplasia. The immunology of infectious disease teaches that the clearance of a pathogenic challenge requires the initiation of an immune response of the appropriate quality and quantity. Clinical experience demonstrates the perils of an inappropriate immune reaction. Pathogens may be of an intracellular (viral, some bacterial strains) or extracellular (bacterial, parasitic) nature; accordingly, the specific immune response is bifurcated into a cell-mediated branch, which offers protection against intracellular pathogens, and an antibody-mediated branch, which offers protection against extracellular pathogens. Following a challenge with Mycobacterium leprae , the leprosy inducing bacterium, a cell-mediated immune response will result in protection whereas an antibody-mediated immune response will result in cachexia and disease progression [ 3 ]. The ability of the immune system to act as a double-edged sword implies that in any given condition the initiation of an immune response may result in either protection or destruction of healthy tissue. An appreciation of the duality of the immune reaction is imperative in the design of immunotherapeutic approaches that attempt to attain a therapeutic benefit through the manipulation of the immune system. Two distinct theories aim to define the role of the immune system in cancer. The theory of immunosurveillance postulates that the role of the immune response in cancer is one of protection – the organism is patrolled for incipient tumor cells by the effector cells of the immune system. In contrast, the theory of immunostimulation postulates that, while in experimental systems highly immunogenic tumors may be eradicated by the immune response, the role of the immune response in spontaneous neoplasia is not one of protection, but rather of tumor promotion. This review gives a historical overview of these theories and highlights recent data supporting the validity of both immunosurveillance and immunostimulation. To explain the conflicting roles of the immune response in neoplasia it must be noted that the immune system is not a single entity, but a complex system of constituents. While these theories have historically been considered to be mutually exclusive, it is proposed that these theories describe the activation of different constituents of the immune system and hence illustrate that an appropriate immune reaction will result in protection whereas an inappropriate immune reaction will result in tumor promotion. The theory of immunosurveillance The rise of the theory of immunosurveillance As early as 1909 Paul Ehrlich postulated that cancer occurs spontaneously in vivo and that the immune system is able to both recognize and protect against it [ 4 ]. In the late 1950s Lewis Thomas [ 5 ] introduced the theory of immunosurveillance, which was subsequently developed by Sir MacFarlane Burnet [ 6 ]. The theory postulates that effector cells of the immune system actively patrol the body to identify and eradicate incipient tumor cells. Following the identification of T cells in the 1970s, these became the effector cells postulated to mediate immunosurveillance. The concept of immunosurveillance initially had much intellectual appeal. First, it could explain clinical observations of spontaneous remission. Second, the most potent chemical carcinogens, dimethlybenzanthracene [ 7 ], urethan [ 8 ] and other polycyclic hydrocarbons [ 9 ], are also powerful immunosuppressors, their suppressive effects being apparent even after a single exposure [ 7 ]. Third, specific immune responses had been observed in the transplantation of chemically induced tumors. Finally, the theory of immunosurveillance was developed at a time when the only known function of T cells was to reject foreign grafts; thus, there was a clear need to further determine the biological function of these cells. Discrepancies in the theory of immunosurveillance A number of discrepancies were noted in the theory of immunosurveillance as initially described by Thomas and Burnet, where T-cells function as the effector cells mediating immunosurveillance. First, T-cell deficient (athymic nude) mice did not develop significantly more cancers than control mice [ 10 ] Second, a corollary of the theory of immunosurveillance is that patients who lack an immune response would have an increased incidence of neoplasia. While this has been reported in immunosuppressed patients who have undergone renal transplant, most of these tumors (as many as 60%) were reported to be hematological malignancies or neoplasms with viral etiology. It would be reasonable to expect the immune system to protect against viral carcinogenesis, as the protective role of the immune system against viral pathogens is well established. One interpretation of these results is that the predisposition toward hematological malignancy is due to the viral etiology of certain lymphomas (which are thought to be caused by the Epstein-Barr virus). The only non-lymphoid tumors that increase significantly upon immunosuppression are non-melanoma skin cancers, cervical carcinoma and Kaposi's sarcoma (the latter two having a viral etiology, specifically the human papilloma virus and human herpes virus 8, respectively). In these early studies, the incidence of most tumor types was not increased by immunosuppression [ 11 ]. In fact, the incidence of mammary carcinomas actually decreases in immunosuppressed individuals [ 12 ]. A third discrepancy is that diseases such as leprosy, sarcoidosis and uremia, which are characterized by immunosuppression, are not accompanied by an increased incidence of tumors [ 13 , 14 ]. Finally, the theory of immunosurveillance predicts that sites that are excluded from the immune system – that is, sites of immunological privilege such as the anterior chamber of the eye and the brain – should have an increased incidence of cancer. However, this is not the case. Furthermore, several studies suggest that incipient tumors may not be immunogenic. A correlation between the dose of carcinogen administrated and the immunogenicity of the subsequent tumor has been reported in mouse models [ 15 - 17 ]. Since human tumors most likely occur as a result of low exposure to carcinogens, incipient tumors would be expected to be poorly immunogenic. In addition, since incipient tumors would contain few cells, the question arises of whether the antigenic load would be sufficient to induce a response [ 18 ]. This phenomenon may be observed in the immunology of infectious diseases where naïve C57Bl/6 mice challenged with Taenia taeniaeformis will reject a large inoculum of the cestode larva yet will succumb to infection by minute inoculations [ 19 ]. Finally, it is only in recently years that the tumor specific- and not tumor associated antigens have been identified [ 20 ]. The revivification of immunosurveillance Over the past two decades, data have emerged suggesting that constituents of the immune system such as natural killer (NK) cells and cytokine networks may be able to guard against cancer. Furthermore, recent studies of the incidence of neoplasia in immunosuppressed patients after organ transplantation, in contrast to earlier studies, reported that these patients are more susceptible to a wide range of cancers, including epithelial cancers [ 21 , 22 ]. Natural killer cells Several studies suggest that NK cells are able to protect against tumors. Beige, natural-killer-cell defective, mice have an increased incidence of spontaneous tumors [ 23 ] and cancer metastases [ 24 - 28 ]. This is consistent with the pattern seen in patients with Chediak Higashi syndrome, an autosomal recessive disorder characterised by abnormal NK cytotoxic function. These patients also have a 200-fold increased risk of developing malignancy [ 29 ]. Furthermore, several studies show that cancer cells secrete soluble factors that suppress NK cells in vitro [ 30 - 32 ] and that patients with a variety of tumor types have suppressed NK cell activity [ 33 - 38 ]. NK cell activity is also a positive prognostic indicator in several tumor types [ 39 - 43 ]. These data suggest that NK cells are involved, directly or indirectly, in the surveillance of incipient tumors and micrometastases. This theory is further substantiated by data showing that oncogene-transfected fibroblasts can be selectively lysed by NK cells while sparing untransfected controls [ 44 ]. The precise mechanism though which NK cells mediated immunosurveillance is not yet understood. It is likely that the role of NK cells, in addition to their direct cytotoxic effects, is to activate other cells of the immune system through providing cytokine support [ 45 ]. Mature NK cells do not produce T-helper 2 (Th2) cytokines, but rather the T-helper1 (Th1) cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ and GM-CSF [ 46 ]. In fact, secretion of IFN-γ by NK cells can influence the development of a Th1 type immune response both against pathogenic agents and against MCA-induced tumors [ 47 ]. Cytokine networks The initiation of an immune response requires cytokine support provided by T-helper cells. The nuance of cytokines produced by these cells determines the type of immune response initiated. The initiation of a cell-mediated immune response requires the support of Th1 cytokines, while the initiation of an antibody-mediated immune response requires the support of Th2 cytokines. T-helper cells that have differentiated into Th1 cells secrete IFN-γ and to a lesser extent interleukin (IL)-2 and IL-12, whereas Th2 cells secrete IL-10, IL-4 and to a lesser extent IL-5. The cell-mediated immune response is generally regarded as possessing tumor-inhibitory activities both clinically and in animal models [ 48 - 51 ]. Accordingly, a number of studies suggest that the expression of Th1 cytokines is associated with a favorable clinical outcome, while the expression of Th2 cytokines is associated with an unfavorable clinical outcome. In renal-cell carcinoma, the presence of an IL-4 receptor polymorphism that resulted in an increase in IL-4 signaling and an increased likelihood of a Th2 response was an independent indicator of adverse prognosis [ 52 ]. In other studies of patients with renal cancer, an elevated level of IL-10 was an adverse prognostic indicator [ 53 ], while the serum levels of IFN-γ were negatively correlated with tumor mass [ 54 ]. In malignant melanoma, patients that had an early relapse had lower serum levels of IL-2 and IL-12. Furthermore, decreases in the serum concentrations of IL-2 and IL-12 and increases in IL-10 were observed at least one month before relapse [ 55 ]. In B-cell diffuse large cell lymphoma, patients who achieved complete remission had a higher ratio of Th1 to Th2 cells [ 56 ]. Finally, in a range of advanced solid cancers, the serum IL-10 level was an independent prognostic indicator of overall survival and time to treatment failure [ 57 ]. These data suggest that the Th1-Th2 paradigm is relevant to cancer, although it should be noted that the Th1/Th2 paradigm is a generalization and that these data show a correlation, and as such are not evidence of a causal relationship. Since many tumors secrete IL-10, it is possible, for example, that the relationship between expression of IL-10 and tumor prognosis reflects the poor prognosis of patients with a larger tumor burden. This is plausible given that the role of IL-10 in tumor immunology is complex (reviewed in [ 58 ]). IL-10 appears to be a pleotropic cytokine whose reported effect is a function of the nuances of the experimental system. While several reports suggest that IL-10 is immunosuppressive, several groups cite IL-10 to have stimulatory effects on T cells and NK cells [ 59 ]. In addition, IL-10 transfected into murine carcinoma models decreases tumorigenicity and sensitizes tumor cells to immune mediated lysis by either NK cells or NK cells and cytotoxic T-cells [ 60 - 62 ]. Clinical data further corroborates a protective role for IL-10 in tumor immunology by showing that metastatic melanoma patients who responded to an immunotherapeutic regimen had tumors with significantly higher expression of IL-10 mRNA as compared to patients that did not respond [ 63 , 64 ]. Furthermore, tumors have a number of specific and non-specific ways of evading a Th1 response. Tumors secrete a number of agents, including transforming growth factor (TGF)-β, IL-10 and prostaglandin E-2, which have been shown to promote a Th2 immune response while suppressing the Th1 immune response. Indeed, it has been shown that the cytokine networks of some cancer patients are skewed toward Th2 [ 55 , 65 , 66 ]. That is, these patients exhibit enhanced expression of Th2 cytokines or decreased expression of Th1 cytokines systemically or in the local tumor microenvironment. The observation that tumors have developed numerous methods of evading the Th-1 response is consistent with the notion of immunosurveillance. Nowell's clonal-evolution hypothesis postulates that cancer is a Darwinian process: mutations that provide a growth or survival advantage will be selected for in the population [ 67 ]. The fact that malignancies have many ways of evading the Th-1 response suggests that the ability to evade this response confers a survival advantage on malignant cells, which further suggests that the Th-1 response poses a threat to the neoplasm. Interferon-γ The Th-1 cytokine IFN-γ has both direct and indirect antitumor properties. A series of experiments have demonstrated the importance of IFN-γ in eradicating incipient tumors by showing an increase in the efficacy of carcinogenesis in the absence of IFN-γ. These experiments were conducted either with neutralizing antibodies to IFN-γ or in animal models deficient in IFN-γ, the IFN-γ receptor or downstream signaling mechanisms of IFN γ, specifically, the signal transducer and activator of transcription-1 protein. In all of these models, an increase in the incidence of MCA-induced tumors and a decrease in the latency period of the tumors were observed [ 68 - 71 ]. Furthermore, an increase in the number of spontaneous lymphomas and lung tumors was observed in IFN-γ deficient mice compared with genetically matched wild-type controls [ 72 ]. IFN-γ may inhibit tumor growth by affecting proliferation, apoptosis and angiogenesis. IFN-γ has been shown to have a direct anti-proliferative effect on certain tumor models [ 73 - 75 ]. It is thought that this effect is mediated through p21 (WAF1/CIP1) and p27 (kip1) as IFN-γ activates these tumor suppressors [ 76 , 77 ]. Furthermore, IFN-γ has been reported to modulate apoptosis in certain models [ 78 , 79 ] by inducing expression of caspase 1 and Fas/FasL [ 80 , 81 ]. With respect to angiogenesis, IFN-γ induces expression of three angiostatic non-ELR (non-Glu-Leu-Arg) chemokines: interferon-gamma-inducible protein 10 (IP-10), monokine induced by gamma interferon and interferon-inducible T-cell alpha chemoattractant [ 82 - 84 ]. The effects of these chemokines on angiogenesis will be discussed. IFN-γ may also have indirect antitumor effects by stimulating an effective antitumor immune response. In addition to influencing the Th1-Th2 cytokine balance, IFN-γ can activate cytotoxic macrophages, NK cells and NK T cells [ 85 ]. The theory of immunostimulation The proof of the principle that an inappropriate type of immune response will enhance tumor growth was demonstrated as early as 1907 by Flexner and Jobling, who showed that injection of dead autologous tumor cells enhanced the growth of pre-existing tumors [ 86 ]. In general, Th2-driven antibody responses to tumors are non-protective and may contribute to tumor progression by inhibiting the Th1 cell-mediated immune response [ 87 ]. However, the notion that the antibody-mediated immune response may be detrimental in cancer was suggested long before Mossman and Coffman demonstrated the Th1-Th2 paradigm in 1986 [ 88 ]. In the 1950s Kaliss popularized the term "immunological enhancement" to describe the enhancement of tumor growth by non-cytotoxic antibodies [ 89 ]. It was theorized that these antibodies bind to tumor cells, masking their epitopes and thus preventing a cell-mediated immune response, although this has never been demonstrated experimentally. In 1972, Richmond Prehn formulated the theory of immunostimulation of tumor growth [ 90 ]. This theory states that, in contrast to the strong immune response generated by transplantable tumors, a quantitatively mild immune response, such as that generated by spontaneous tumors, is stimulatory to the growth of neoplasia. Several experimental observations support the hypothesis that such a weak immune response to cancer may stimulate tumor growth. The co-injection of lymphocytes (spleen cells) from syngeneic mice that had been growing tumors for 10–20 days with tumor cells from MCA-induced sarcomas into thymectomized irradiated syngeneic mice at a range of doses accelerated tumor growth when the ratio of lymphocytes to tumor cells was low [ 90 ]. However, when the ratio of lymphocytes to tumor cells was high, lymphocytes from specifically immunized mice inhibited growth compared with naïve lymphocytes that continued to augment tumor growth. This suggests the existence of a biphasic dose response whereas a "weak" immune response results in stimulation of tumor growth while a strong immune response results in protection. This premise is also demonstrated in another study where MCA induction of tumors occurred more rapidly in irradiated thymectomized mice that had received increasing doses of lymphocytes (spleen cells) to partially restore their immune system than in mice that had either fully restored or unrestored immune systems [ 91 ]. Comparable results have been observed in mice that have had their immune systems compromised to various degrees by irradiation [ 92 ] and in T-cell deficient nude mice that have had their immune systems partially restored by the injection of various quantities of thymic or spleen cells [ 93 ]. These studies support the notion that a weak antitumor immune response will not confer protection and may exacerbate tumor growth. Furthermore, in transplantation immunology, an immune response against a graft generally promotes graft rejection. However, rabbits that received skin grafts of similar but not identical major histocompatibility complex launched an immune response that resulted not in rejection but in enhanced growth of the tissue, resulting in hyperplasia [ 94 ]. This is consistent with the notion that a quantitatively "weak" immune response against a tumor may enhance tumor growth. Collectively, these data support the essence of the theory of immunostimulation: specifically, that spontaneous tumors may not stimulate an appropriate immune response but rather stimulate non-protective immune responses that quantitatively are not adequate for tumor eradication. These resulting "weak" immune responses are not merely non-protective but actually facilitate tumor growth. Specifically, a "weak" immune response is a state in which immunological recognition of the tumor occurs but resolution is not achieved. As this theory was developed in an era when our understanding of the composition of the immune system was limited, the exact mechanism by which a "weak" immune response stimulates tumor growth has yet to be defined in the terminology of contemporary immunology. Recent data, however, suggest numerous mechanisms by which the immune system can facilitate tumor growth and progression. Inappropriate immune reactions exacerbate cancer Based on the observation that a tumor is in a state of chronic inflammation, Dvorak compared cancer to a wound that never heals [ 95 ]. Numerous tumor types constitutively produce cytokines and chemokines; this results in the migration of leukocytes into tumors. Unfortunately, these immune infiltrates often do not offer protection but actually facilitate tumor growth. Tumor-infiltrating leukocytes can facilitate tumor progression by secreting growth factors, reactive oxygen and nitrogen species, proteases, prostaglandins and angiogenic growth factors. Reactive oxygen and nitrogen species may directly promote tumor progression by inducing DNA damage, and hence the acquisition of additional mutations. In addition, these cells may skew the cytokine milieu to favor the generation of a non-protective Th2 immune response. Mast cells and macrophages are examples of inflammatory cells that are often recruited to tumor sites through chemotactic gradients. The mechanisms by which mast cells, macrophages and chemokines themselves facilitate tumor growth and progression are discussed below. Mast cells Mast cells are leukocytes that contain inflammatory agents, such as proteases, histamine and heparin, and mediate hypersensitivity reactions. The possibility that mast cells are involved in cancer has been considered since the late 1800s. In 1877, Paul Ehrlich described a mast cell for the first time in his doctoral thesis, after identifying it using histological staining. In subsequent investigations, he and others observed that mast cells localize around tumor tissues [ 96 ]. Interestingly, mast cells were more likely to be localized in the periphery than in the centre of tumor sections. Many types of human tumors contain mast-cell infiltrates [ 97 - 100 ]. The accumulation of mast cells in neoplastic tissue results from the secretion of growth factors such as vascular endothelial growth factor (VEGF) [ 101 ], epidermal growth factor [ 102 ], basic fibroblast growth factor (bFGF) [ 103 , 104 ], platelet derived growth factor [ 101 ] and stem cell factor (SCF) [ 105 ] by the tumor. These cytokines act as chemotactic agents to induce the migration of mast cells. A number of studies show a correlation between mast-cell infiltration and tumor progression. For example, co-injection of mast cells with an inoculum of rat sarcoma tumors results in enhanced tumor growth, while pharmacologically decreasing the quantity of mast cells slows tumor growth [ 106 ]. Furthermore, genetic evidence supports the role of mast cells in tumor growth and progression. SCF is important for mast-cell development, proliferation, migration and degranulation. W/W v mice (which express a mutation in the SCF receptor) do not develop functional mast cells. In W/W v mice, tumors do not metastasize as readily and they vascularize at a slower rate than in wild-type mice. Tumor metastasis and vascularization return to wild-type levels following restoration of mast cells [ 107 ]. Furthermore, administering antisense targeting SCF in a rat mammary-tumor model results in a decrease in mast-cell degranulation, microvascular density and tumor growth [ 108 ]. These studies suggest that the presence of mast cells in tumors is not indicative of a protective immune reaction, as these cells actually facilitate tumor progression. The mechanisms by which mast cells facilitate tumor progression appear to involve the factors contained within mast-cell granules. Experimental evidence suggests that degranulation is critical in the ability of mast cells to enhance tumor growth. Inhibition of mast-cell degranulation using disodium cromoglycate impedes tumor growth [ 109 ]. One of the functions of degranulation in tumor growth may be to facilitate angiogenesis. Addition of mast cells or isolated mast-cell granules induces vascularization in the chorioallantoic membrane model, while no effect of adding previously degranulated mast cells was observed [ 110 ]. This effect appears to be partially mediated through bFGF and VEGF, as addition of anti-bFGF and anti-VEGF antibodies significantly decreased the degree of vascularization. Indeed, mast cells secrete a number of growth factors that regulate angiogenesis and endothelial-cell survival, including TNF-α [ 111 ], IL-8 [ 111 ], bFGF [ 112 ] and VEGF [ 113 , 114 ]. Moreover, mast-cell granules contain the proteolytic enzyme tryptase, while some mast-cell subsets also contain chymase. These enzymes have both direct and indirect effects on the integrity of the extracellular matrix (ECM). Directly, these enzymes induce degradation of ECM components [ 115 ]. Indirectly, they can induce degradation of the ECM by activating latent forms of matrix metalloproteinases [ 116 ]. Furthermore, it has been reported that these proteinases may induce proliferation of vascular endothelial cells [ 117 ]. As is often the case in networks, activation of one component induces the activation or suppression of interacting components. Activated mast cells secrete a number of cytokines that induce the chemotactic migration of macrophages into the tumor, including IL-1, IL-6, TNF-α, IL-8, monocyte chemotactic protein-1, macrophage inflammatory protein-1α and macrophage inflammatory protein-1β [ 118 , 119 ]. The effect of macrophages on tumor growth and progression is discussed in the following section. In addition, activation of mast cells induces the synthesis of the cylooxygenase enzyme, which is involved in the metabolism of arachidonic acid [ 120 - 122 ] and prostaglandins. Pharmacological inhibition of cylooxygenase significantly impedes metastasis in several animal models [ 123 - 125 ]. Macrophages Macrophage infiltration of tumor sites has been observed in a range of tumor types. Monocytes are recruited to the tumor by constitutive tumor-cell expression of chemoattractant cytokines. In fact, the level of expression of monocyte chemotactic protein-1, macrophage colony stimulating factor (M-CSF) and VEGF by tumor cells correlates well with the extent of macrophage infiltration [ 126 , 127 ]. Within the tumor microenvironment, monocytes differentiate into macrophages. The role of the macrophage in the tumor microenvironment appears to be specific to the tumor type. In breast [ 128 ], cervical [ 129 ] and bladder [ 130 ] cancers macrophage infiltration is an adverse prognostic indicator. However, in prostate [ 131 , 132 ], lung [ 133 , 134 ] and brain [ 135 , 136 ] cancers the prognostic significance of tumor associated macrophages, TAMs, depends on the method of assessing macrophage infiltration, the endpoints of the study and the specifics of the patient cohort. This discrepancy highlights the chameleon-like nature of the macrophage. Depending on their microenvironment, macrophages may either exhibit antitumor cytotoxic activity or facilitate tumor growth and progression while reinforcing a Th2 biased immune response [ 137 ]. Following exposure to IFN-γ or bacterial lipopolysaccharide, macrophages can exhibit direct or indirect tumor cytotoxicity. Macrophages may participate in antibody-dependent cellular cytotoxicity responses if an IgG2a antibody binds to a tumor surface antigen. In such a scenario, the macrophage would bind to the Fc portion of the antibody, releasing cytotoxic mediators such as proteinases and TNF-α. Macrophages may also mediate cytotoxicity independently of antibodies through the secretion of reactive oxygen and nitrogen species, proteinases and TNF-α. Furthermore, macrophages of this "cytotoxic" phenotype affect the cytokine profile of the microenvironment because they secrete IL-12. IL-12 favors the differentiation of naïve T-helper cells to Th1 cells, which will subsequently secrete IFN-γ and TNF-β. The role of these cytokines in the promotion of a cell-mediated immune response has already been discussed. Finally, cytotoxic macrophages produce matrix metalloproteinase-12 [ 138 ]. Matrix metalloproteinase-12 has been shown to convert plasminogen to angiostatin [ 139 ]. Angiostatin is important in the inhibition of angiogenesis [ 140 ]. In contrast to macrophages that are activated by classical mechanisms and are capable of cytotoxic activity, TAMs, for the most part, do not exhibit cytotoxic activity, are Th1 immunosuppressive and, in fact, facilitate tumor growth, vascularization and metastasis. Tumors secrete a number of cytokines including IL-4, IL-10, TGF-β, prostaglandin E-2 and VEGF. These factors modulate the macrophage phenotype, changing them from cytotoxic macrophages to suppressive macrophages [ 141 - 143 ]. Furthermore, TAMs appear to have functional defects; for example, in contrast to other macrophages, TAMs are poor antigen-presenting cells [ 141 ] and have reduced cytotoxicity owing to impaired production of TNF-α [ 144 ] and nitric oxide [ 145 ]. However, these impairments in the function of TAMs are consistent with macrophages that are not activated rather than defective. A number of studies suggest that TAMs facilitate tumor growth, vascularization and metastases. As previously stated, numerous studies have shown the presence of TAMs to be an adverse prognostic indicator. In addition, genetic evidence suggests that macrophages are important in vascularization and metastasis of experimental tumors. To assess the role of macrophages in carcinogenesis, the M-CSF deficient osteoporotic (op/op) mouse was crossed with the polyoma virus middle T transgenic mouse, which develops spontaneous mammary tumors. While no differences were observed in the early stages of tumor growth, the resulting offspring of these tumor-bearing mice had a reduced rate of progression to invasive carcinoma and contained fewer pulmonary metastases than control mice [ 146 ]. Furthermore, induced M-CSF expression in the mammary tissue of these mice resulted in restored metastatic spread of experimental tumors. In addition, transplantation of the Lewis lung carcinoma in the op/op mouse resulted in a decrease in its growth and vascularization compared with wild-type littermates [ 147 ]. This attenuation could be reversed by administration of M-CSF. These data suggest that macrophage infiltration is important in angiogenesis and tumor progression. Interestingly, TAMs are not found ubiquitously within tumor tissue. Data suggest that TAMs localize to poorly vascularized regions – that is, regions characterized by hypoxia, low pH and tissue necrosis [ 127 , 148 - 150 ]. In response to hypoxia, macrophages express a number of hypoxia-regulated gene-products, including VEGF, hypoxia inducible factor 1α and hypoxia inducible factor 2α [ 149 , 151 , 152 ]. Furthermore, hypoxic conditions impair the chemotactic migration of macrophages [ 150 , 153 ]. This suggests that macrophages could be detained in regions of low oxygen tension. While the precise nature of the relationship between macrophages and hypoxia requires further investigation (macrophages are phagocytic cells, so their physiological role may well be to engulf necrotic tissues), it is possible that macrophage recruitment to these sites facilitates angiogenesis. One mechanism by which TAMs may augment angiogenesis is through the production of a plethora of cytokines and proteinases. As mentioned above, hypoxia induces production of VEGF. The angiogenic properties of VEGF have been extensively discussed by others [ 154 ]. In addition to VEGF, the repertoire of cytokines produced by TAMs includes granulocyte macrophage colony-stimulating factor, TGF-α, TGF-β, IL-1, IL-6, IL-8 and prostaglandin E-2. These cytokines are involved in the regulation of angiogenesis. Furthermore, TAMs secrete urokinase-type plasminogen activator and matrix metalloproteinase-9 [ 155 , 156 ]. These proteinases are involved in the induction of angiogenesis and metastasis through degradation of the ECM. Finally, TAMs have numerous indirect immunological effects. TAMs produce copious amounts of IL-10 but low amounts of IL-12 [ 157 ]. This shift in the cytokine profile reinforces the Th2 imbalance that is often present in cancer models. More significantly, IL-10 prevents T-cell activation by inducing a state of anergy (a state of T-cell non-responsiveness associated with tolerance induction). IL-10 can induce anergy both in T cells that have been activated in its presence and in T-cells activated by antigen-presenting cells that were previously exposed to IL-10 [ 158 ]. These data suggest that TAMs may also facilitate tumor growth by inducing tolerance to the tumor and hence suppressing the antitumor immune response. Chemokines Chemokines are a subclass of cytokines that direct the migration of leukocytes to sites of inflammation. It has recently been observed that tumors express chemokine receptors [ 159 - 161 ]. Thus, it should not come as a surprise that, aside from their role in mediating the recruitment of tumor-infiltrating leukocytes to tumor sites, chemokines may also affect neoplastic proliferation, neovascularization and metastasis. Specific chemokines, such as growth-related oncogene (GRO)-α, GRO-β, GRO-γ and IL-8, directly induce the proliferation of melanoma cells [ 162 ], while ligands of the chemokine receptor CXCR-2, such as IL-8, induce proliferation of lung [ 163 , 164 ], ovarian [ 165 ], pancreatic [ 166 ] and head and neck [ 167 ] tumors. Chemokines may both augment and inhibit angiogenesis. In general, chemokines with an ELR motif promote angiogenesis and facilitate the chemotactic migration of endothelial cells [ 168 ]. Nevertheless, IFN-γ induces the expression of three non-ELR chemokines that have been reported to have angiostatic properties. For example, expression of IP-10 is negatively correlated with human lung-cancer tumor growth [ 169 ], while administering recombinant IP-10 slows tumor growth [ 170 ]. Furthermore, administration of IL-12 inhibits bFGF-mediated in vivo neovascularization of matrigel [ 171 ]. The angiostatic effects of IL-12 appear to be mediated through the induction of IP-10 and of monokine induced by gamma interferon, as neutralizing antibodies to these chemokines negated the angiostatic effects of IL-12 [ 172 , 173 ]. Finally, in some systems IL-8 activates the transcription of matrix metalloproteinase and is associated with an increased degree of invasion and metastasis. Furthermore, chemokines may be involved in the attraction of circulating cancer cells to sites of metastasis [ 174 - 177 ]. Strategic research directions Based on our knowledge of tumor immune evasion and immune contribution to growth of tumors, several therapeutic implications arise including: a) Use of adjuvant therapies to decrease tumor immune suppression while vaccination, b) Extracorporeal elimination of immune suppressive molecules; and c) Gene silencing of tumors to generate immunity. The armamentarium of clinically useful drugs for inhibition of tumor immunity is rapidly growing. The challenge is to identify drugs that intrinsically possess antitumor activity, while at the same time can reverse immune stimulation. One promising candidate that fits these criteria is the clinically used anti-VEGF antibody bevacizumab (Avastin). Currently approved by the FDA for treatment of advanced colon cancer patients, this antibody is believed to mediate its effects primarily by inhibition of angiogenesis [ 178 ]. From the tumor immunotherapy perspective, VEGF plays an important role for tumor suppression of immune responses. In fact, administration of anti-VEGF antibody increases efficacy of immunotherapy in mouse models [ 179 ]. Mechanisms by which VEGF inhibits antitumor immunity include suppression of NF-kB activation in DC resulting in an immature phenotype [ 180 ], as well as suppression of T cell activation [ 181 ]. Suppression of immune inhibitory molecules could also be accomplished by immunization. For example, the pregnancy associated molecule human chorionic gonadotropin (hCG) is associated with inhibition of Th1 generation in vitro as well as in vivo [ 182 ]. Vaccination with the carboxy-terminal peptide of hCG has been shown to break tolerance to this self-molecule and induce a marginal anticancer response [ 183 ]. Combining vaccination against immune suppressive molecules with vaccination against tumor-specific antigens will likely improve efficacy. Another method of reversing tumor associated immune suppression would involve extracorporeal removal of inhibitory molecules. Although plasmapheresis techniques have been attempted with mediocre success [ 184 ], a novel and promising method involves ultrapheresis. Lentz et al described a pilot study of 16 metastatic patients in which the <100,000 kDa fraction was removed through membrane ultrapheresis, Six of the 16 patients had reduction of the sum of mean cross-sectional diameters of measureable lesions by 50% or more. Additionally, tumor infiltrating lymphocytes were observed in many of the lesions after 2 months of treatment [ 185 ]. Follow-up studies demonstrated that the immune suppressive component being removed through the ultrapheresis procedure was soluble TNF-R alpha [ 186 ]. Recent developments in hollow-fiber technologies allow for specific removal of plasma bourne components such as HIV gp120 and various toxins [ 187 ]. Applications of these techniques to immune therapy could yield valuable new approaches that are currently not studied. The introduction of RNA interference (RNAi) as a potent method of gene-specific silencing has opened new territories for gene therapy. Successful use of RNAi for immune modulation was first reported by Hill et al [ 188 ]. Subsequently, it was demonstrated that silencing of the inhibitory cytokine IL-10 led to the generation of dendritic cells with potent Th1 priming abilities [ 189 ]. More recently, siRNA modified DC were used for induction of antitumor immunity [ 190 ]. The fact that naked siRNA can be directly endocytosed by target cells [ 191 ], or administered using polyethylenimine-complexe [ 192 ], suggests the possibility of direct intratumoral injection of siRNA targeting immune suppressive molecules. Additionally, tumor-targeting immunoliposomes could be used for systemic delivery of siRNA into cancer cells. Suitable targets would include the wide variety of immune suppressive molecules mentioned above. Conclusions The debate on the role of the immune system in cancer has been one of the most controversial areas of science with opinions vacillating from optimistic highs to skeptic nadirs on a cyclical basis. These oscillations in opinion that are reflective of the relative progress in the immunotherapy of cancer, can be explained by the complexity of the relationship of the immune system and cancer – an interaction that can result in anti-tumor protection, tumor promotion, or in no net effect. The apparent schizoid role of the immune system in cancer may in fact be a variation on a theme borrowed from the immunology of infectious disease. That is, the mere generation of an immune response is not sufficient to attain protection against a pathogen: it is only an immune response of the proper type and the proper magnitude that will result in protection. An immune response against an agent that is not of the proper type and magnitude will be deleterious to the host. A proper type of immune response against cancer will result in protection. Evidence exists that in set conditions components of the immune system can recognize and eradicate incipient cancer cells. It is likely that some sort of immune surveillance exists in the healthy individual – though the exact mechanisms have yet to be elucidated. The genetic evidence discussed herein suggests that NK cells and IFN-γ are likely to be involved in this protection. Nevertheless, the clinical presentation of cancer suggests that neoplasia can evade these putative mechanisms of immunosurveillance. This further suggests that the ensuing immune response is not protective, or not adequately protective, to control the nascent tumor. However, this lack of a protective response is not the whole extent of the role of the immune system in cancer. Several examples have been presented in which the immune response actually facilitates tumor growth, neovascularization and progression. These observations suggest a mechanism that may explain the theory of immunostimulation, though undoubtedly, many other mechanisms may exist. This duality of the immune system in cancer arises in part because of the decentralized nature of the immune system. The non-protective immune reaction that arises resembles a bureaucratic organization in which a lack of orchestration between departments results in redundancy and counter-productivity: the individual entities that constitute the immune system react to the presence of the tumor yet they are not orchestrated to achieve the end point of tumor eradication. Secondly, in advanced tumors, the duality of the immune system in cancer may also arise from the bilateral nature of the interaction between the tumor and the immune mechanisms of the host. Not only can the immune system affect the tumor, but the tumor may also affect the immune system. The skewed cytokine milieu of a tumor, which results in the recruitment of inflammatory cells through the secretion of chemokines and growth factors and which further prevents the cytotoxic activation of these cells, is an example of how the tumor influences the immune system. This effect of the tumor on the immune system may be explained by the theory of immunoediting, which describes the iterative process of selection for cells that can evade the natural immunosurveillance mechanisms [ 193 ]. This selection process may generate tumors that not only escape detection by the immune system, but that have actually generated mechanisms of depressing those branches of the immune response that would offer anti-tumor protection and/or enhancing those branches that would elicit tumor promotion. Akin to the successful development of vaccines against infectious agents, the development of effective immunotherapy against cancer will require recognition of the duality of the immune response in cancer, and the stimulation of the proper type of immune response. The challenges ahead, especially in late-stage patients who have undergone immunoediting, will be to overcome the tendencies of the tumor to elicit an inappropriate immune response, one used by the tumor to promote its own growth. It may be the case that in subsets of patients the sort of immunotherapy that is desirable is not that which stimulates the immune system but that which suppresses an inappropriate, tumor promoting, immune reaction [ 194 , 195 ]. Chemotherapy, may in fact be an example of 'immunotherapy'. Chemotherapy both directly and indirectly affects the immune system; however, given the multiplicity of the functions of the immune system in cancer, the net effect of this treatment modality remains to be defined. While the direct effect of chemotherapy may be myelosuppression, this may not be an undesirable consequence given that cells of the myeloid lineage, such as mast cells and macrophages, often promote the growth, vascularization and invasion of tumors. Furthermore, myelosuppression may actually stimulate a protective, Th1-type, cell mediated immune response because mast cells and macrophages secrete factors that promote a Th2 type of immune response. "If only it were all so simple." If only the immune system were all good and it were necessary only to stimulate it to achieve the eradication of tumors. But the potential for good and evil is entwined within the heart of the immune reaction, and who is willing to destroy their own heart? | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549049.xml |
449908 | Remembering Which X Chromosome to Use | null | In mammals, males usually have one X and one Y chromosome and females have two X chromosomes. This crucial difference sets the sexes apart, but also creates a problem—female cells have the potential to turn out twice as much X-based gene product as necessary. Males with multiple–X chromosome syndromes face a similar problem. As if to avoid an overdose of X-related proteins, cells in the early embryo inactivate all but one X chromosome. The choice of which X (or Xs) to inactivate is apparently random, but once made, it persists across cell divisions and the specializations that determine a cell's ultimate fate, or type. Chromosome-wide histone H3 lysine 27 trimethylation caused by Xist expression A gene called Xist —which resides on the X chromosome—has a central role in X chromosome inactivation. It creates a special RNA molecule that spreads from its point of production down the length of the X chromosome, repressing its genes and inactivating the chromosome. After about one cell cycle, this gene silencing no longer requires Xist RNA. Daughter cells somehow remember which X to keep mute. In this month's PLoS Biology , geneticist Anton Wutz and colleagues at the Research Institute of Molecular Pathology in Vienna show that Xist expression during a critical period very early in embryonic development creates a chromosomal memory, independent of X silencing, that might help maintain X inactivation across cell generations. The molecular underpinnings of X inactivation seem to center on histones, the protein spools around which DNA coils its length. DNA and histones form complexes called chromatin, which undergoes many structural modifications that have important effects on gene expression. For example, tightly packed chromatin inhibits gene expression in its closely curled segments. Not surprisingly, the inactivated X chromosome is coiled into this dense form, called heterochromatin. In the standing model of X inactivation, the Xist gene mediates alterations to histones (such as the addition of chemical compounds called methyl groups) along the X chromosome, which result in heterochromatin formation. As this structure is passed on to daughter cells, X silencing is perpetuated. To explore the molecular changes that mediate X chromosome inactivation, Wutz and colleagues inserted a special Xist gene into the X chromosome of male mouse embryonic stem cells, so they could turn Xist expression “on” and “off” at will. The stem cells represent the earliest, unspecialized cells of a mouse embryo. Since the cells can be induced to differentiate in culture, they provide the opportunity to study the relationship between differentiation and X chromosome inactivation (which would not normally happen at all in these “male” cells). Using this system, the authors have shown previously that Xist must act during a critical window very early during stem cell differentiation—within the first 24 hours. Wutz and colleagues now show that after that window the X chromosome inactivation can still be reversed, but after an additional 24 hours, it cannot. There appears to be a “memory” of Xist action, which leads to the permanent shutting down of the chromosome. The importance of this observation is that it establishes a new step in the process of X chromosome inactivation—between the action of Xist and the establishment of irreversible silencing. By looking at the kinetics of histone modification, gene silencing, and Xist action, Wutz and colleagues further show that although certain histones are methylated at specific locations during this period in response to Xist , these modifications do not themselves constitute the chromosomal memory. The nature of the memory remains mysterious. Further experiments, perhaps looking at different histone modifications, will be required. Clarification of the events that lead to X inactivation will also improve our knowledge of how changes in the organization and structure of chromosomes can influence the activity of genes. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC449908.xml |
552327 | Replicative Homeostasis: A fundamental mechanism mediating selective viral replication and escape mutation | Hepatitis C (HCV), hepatitis B (HBV), the human immunodeficiency viruses (HIV), and other viruses that replicate via RNA intermediaries, cause an enormous burden of disease and premature death worldwide. These viruses circulate within infected hosts as vast populations of closely related, but genetically diverse, molecules known as "quasispecies". The mechanism(s) by which this extreme genetic and antigenic diversity is stably maintained are unclear, but are fundamental to understanding viral persistence and pathobiology. The persistence of HCV, an RNA virus, is especially problematic and HCV stability, maintained despite rapid genomic mutation, is highly paradoxical. This paper presents the hypothesis, and evidence, that viruses capable of persistent infection autoregulate replication and the likely mechanism mediating autoregulation – Replicative Homeostasis – is described. Replicative homeostasis causes formation of stable, but highly reactive, equilibria that drive quasispecies expansion and generates escape mutation. Replicative homeostasis explains both viral kinetics and the enigma of RNA quasispecies stability and provides a rational, mechanistic basis for all observed viral behaviours and host responses. More importantly, this paradigm has specific therapeutic implication and defines, precisely, new approaches to antiviral therapy. Replicative homeostasis may also modulate cellular gene expression. | Background 1. Disease burden Hepatitis C (HCV), HBV and HIV are major causes of premature death and morbidity globally. These infections are frequently life-long; Hepatitis viruses may result in progressive injury to the liver and cirrhosis, and death from liver failure, or hepatocellular carcinoma, while HIV causes progressive immune depletion and death from the acquired immunodeficiency syndrome (AIDS). Together, these infections cause millions of premature deaths annually, predominantly in "developing" countries. Other viruses replicating via RNA intermediaries cause similar morbidity among domestic and wild animal populations. While education, public health measures and vaccination (for HBV) have resulted in significant progress in disease control, therapy of established viral infection remains unsatisfactory. 2. Viral replication RNA viruses and retroviruses replicate, at least in part, by RNA polymerases (RNA pol ), enzymes that lack either fidelity or proofreading function [ 76 ]. During replication of hepatitis C HCV or HIV each new genome differs from the parental template by up to ten nucleotides [ 61 ] due to RNA pol infidelity that introduces errors at ~1 × 10 -5 mutations / base RNA synthesised. Viruses replicate by copying antigenomic intermediate templates and hence obey exponential growth kinetics, such that [RNA] t = [RNA] (t-1) e k , where [RNA] t is virus concentration at time (t) and k a growth constant. However, because of RNA pol infidelity, wild-type (wt) virus will accumulate at [RNA wt ] t = [RNA wt ] (t-1) •(1-ρ)•K 1 and variant forms (mt) at [RNA mt ] t ≈ ([RNA wt ] (t-1) •ρ + [RNA mt ] (t-1) )•K 1 , where ρ is the probability of mutation during replication and K 1 = e k . Therefore, while wild-type virus predominates early, replication (and intracellular accumulation) of variant virus and viral proteins will accelerate (in a ratio of ([RNA wt ] (t-1) •ρ + [RNA mt ] (t-1) )/ [RNA wt ] (t-1) •(1-ρ) compared to wild type) and variant viral RNAs will rapidly predominate (Figure 1 ). Mutations progressively accumulate in RNA viruses [ 17 ] and ultimately variant RNAs and proteins, if variant RNAs are translated, will become dominant. It is also likely some variant viral proteins will resist cellular trafficking, further accelerating the intracellular accumulation of variant forms relative to wild type. Figure 1 Effect of RNApol fidelity on replication . Each replication cycle may produce either wild-type (Wt) or variant (Mt) copies of parental template in a ratio determined by polymerase fidelity. If HCV RNA pol M u is 10 -5 mutations per base RNA synthesized, Mt:Wt ratio at G 1 is ~9:1, by G 3 unmutated parental genome is 6.8 × 10 -4 of total virus population, and by G 20 7.5 × 10 -22 The paradox of quasispecies stability Two fundamental problems critical to understanding RNA virus quasispecies biology arise because of RNA polymerase infidelity and the mode of viral replication: 1: Replication kinetics Hepatitis C, HIV, and HBV and other viruses, have broadly similar kinetics (Figure 2 ); initial high level viral replication that rapidly declines to relatively constant low-level viraemia [ 11 , 12 ], typically 2–3 logs lower than at peak, for prolonged periods, a kinetic profile attributed to "immune control" [ 12 ]. However, immune control is a conceptually problematic explanation for the initial decline in viral load; For example; why would potent host responses (of whatever type; humoral, cell mediated or intracellular immunity, or any combination thereof), having reduced viral load and antigenic diversity by a factor of 10 2–3 within days, falter once less than 1% of virus remains? Figure 2 Viral kinetic paradox . Viral replication kinetics (—). If host factors (I c , black arrows) reduce viral replication acutely (point A), then they must exceed viral forces (V e , grey arrows). At equilibrium (e.g. points B or C) host forces must balance viral forces; I c must therefore fall by a factor of 10 2–3 from A. Formally 1. Assume immune mechanisms reduce initial viral replication. 2. Let I c(t) represent the immune forces favouring viral clearance and V e(t) viral forces promoting quasispecies expansion pressures at time (t). 3. Assume immune pressures I c required to clear virus are proportional to viral concentration [V], that is; V e ∝ [V] (or V e = k e [V] where k e is some constant), so that I c required to clear one viral particle I c(1) is less than that I c required to clear 10 viral particles Ic (10) . 4. At equilibrium (e.g. time points B or C, Figure. 2 ) immune clearance pressures approximate viral antigenic expansion pressures: I c(b or c) ≈ V e(b or c) . Eq.1 5. If I c causes the reduced viral load seen between time A and time B or C, [V e(a) ] ⇒ [V e(b or c) ], then immune clearance pressures must exceed viral expansion pressures at that time i.e. I c(a) > V e(a) . Eq.2 6. As viral antigenic expansion pressures at time A exceed those at time (B or C) by 10 2–3 [V (a) ] ≈ [V (b or c) ]• 10 2–3 , and I c(b or c) = V e(b or c) then immune clearance pressures at time A exceed those at time (B or C) by10 2–3 I c(a) >I c(b or c) • 10 2–3 . That is, immune pressures fall by 10 2–3 between time A and B or C, (Figure. 2 ). Prompting i) Why, and by what mechanism, would immune forces, or any other host defense mechanisms, fall by 10 2–3 over days between time A and B or C? There is, of course, no evidence immune pressures fall, and very considerable evidence both antibody and adaptive T cell responses are increasing when viral replication is falling [ 5 , 12 ]. These facts are irreconcilable with the notion that immune or other any host mechanisms control initial viral replication and strongly suggest immune or any other host mechanism(s) are not the primary reason viral load falls initially. Further, as down-regulation of viral replication frequently occurs prior to development of neutralising antibody, in the absence of any demonstrable antiviral antibody, or T-cell responses [ 25 , 41 ], and without lysis of infected cells [ 25 ], it is difficult to argue, with any conviction, that either humoral or cellular immune responses primarily cause reduced viral replication. Evidence that prior HCV infection does not confer protective immunity against either heterologous HCV infection in chimpanzee [ 22 ]or either homotypic [ 33 ] or heterotypic [ 32 ] human reinfection further undermines the paradigm of "immune control". Inhibition of immune or other host mechanisms is an untenable explanation of this massive apparent fall in immune clearance pressures; if occurred to any degree, an increase, rather than the observed decrease, in viremia would result. In the absence of a rational host mechanism consistent with observed viral kinetic data, the ineluctable conclusion is that non-host (i.e. viral) mechanisms (i.e. viral auto regulation) must be operative. Chronic viral persistence raises other issues; At steady state (e.g. points B or C, Figure. 2 ), the rate of HIV and HCV production is estimated at 10 10 molecules / day [ 11 , 29 , 52 , 57 ] while HBV production may be 10 11 molecules/day resulting in an average viral load of 10 10 molecules/person [ 52 , 57 ]. However, during peak replication virus production may 10 2–3 times the basal rate [ 11 , 12 ], indicating enormous reserve replicative capacity. As basal viral replication is clearly sufficient for long-term stability, and kinetic analysis suggests viral, rather than host, factors control viral replication, the following questions are posed: When challenged, how do viruses "sense" the threat and by what mechanism do they modulate replication in response? Problem 2: Mutation rate The stability of RNA viral quasispecies poses a major problem: During viral replication the copied genome may either identical to or a variant of parental template (Figure. 1 ). The probability (ρ) of a mutation occurring during replication is a function of polymerase fidelity; During one replication cycle ρ = (1-(1-M μ ) n ), where (M μ ) is mutation rate and (n) genome size. Hepatitis C (a ~9200 bp RNA virus) RNA pol introduces mutation at 10 -5 substitutions/base, ρ≈0.912. However, for multiple (θ) replications cycles, ρ = (1-(1-M μ ) n ) θ . After 20 replication cycles, occurring in <7 days in most patients [ 52 , 57 ], the probability of any original genome remaining un-mutated is ρ o ≈7.5 × 10 -22 , meaning effective loss of sequence information, an outcome that should cause quasispecies extinction [ 16 ]. Persistence of stable RNA viral quasispecies is, therefore, highly paradoxical [ 18 ]. This "theoretical impossibility" of RNA quasispecies stability suggests either a) the consistently reported rates of RNA pol infidelity are incorrect (which, even if true, would only delay quasispecies extinction; if M μ = 10 -10 , ρ o <10 -40 within 100 days etc.) or b) that innate viral mechanism(s) control RNA pol fidelity and mediate selective replication of consensus sequence genomes. Thus, rates of viral mutation are tightly constrained by the necessity to retain sequence information. On the other hand, overly faithful template replication will restrict antigenic diversity, rendering virus susceptible to immune destruction and unresponsive to ongoing cellular changes. The necessity to retain sequence information by adequate replicative fidelity, and the later requirements (in terms of replicase ⇒ RNA pol evolution) of viruses to access cells via evolving cell receptors and evade host defence mechanisms, has placed constraints on replicase (RNA pol ) function that dictate polymerase fidelity must be tightly, and dynamically, controlled (Figure 3a ). Figure 3 a. Constraints on viral mutation . Inadequate polymerase fidelity will cause loss of sequence information and quasispcies extinction (A, B), while inadequate viral mutation will result in immune recognition and viral clearance (D,E). Viral persistence requires polymerase fidelity responsive to the host environment (C). 3b. Constraints on viral replication . Overly rapid replication will cause cell lysis, tissue injury and premature host death (A,B), while inadequate replication will result viral latency or clearance (D,E). Viral persistence with optimal evolutionary stability requires a polymerase responsive to the host environment (C). Evolutionary constraints on viral replication Optimal viral replication is a compromise between maximising host-to-host viral transmission at each host contact versus maximising transmission at sometime during the host's life: Uncontrolled, exponential growth, as might result from the mode of viral replication, would cause rapid cell lysis, host death and a reduced likelihood of stable host-to-host transmission, a prerequisite for viral survival on an evolutionary timescale. While maximising the probability of host-to-host transmission at each contact, high-level viral replication increases the probability of host disease, thus reducing opportunity for transmission long term. Contrariwise, adverse viral outcomes may result from inadequate viral replication causing increased clearance and reduced host-to-host transmission. Viruses that cause premature host death or that are cleared by host mechanisms before transmission to, and infection of, other hosts are biological failures that have strong Darwinian pressures acting against them. Optimal long-term viral stability, therefore, dictates viral replication rates (that is, polymerase processivity) and mutation frequency (that is, polymerase fidelity) must be closely regulated (Figure 3b ). Hypothesis That viruses capable of chronic persistence auto-regulate replication and mutation rates by replicative homeostasis. Replicative homeostasis results when RNA polymerase end-translation products (envelope and contiguously encoded accessory proteins) interact with RNA pol to alter processivity and fidelity. Evidence for Autoregulation Substantial clinical and in-vitro evidence, including the kinetic paradox indicate viruses auto-regulate. During successful antiviral treatment levels of virus fall sharply [ 12 , 29 , 52 , 53 , 57 ], often becoming undetectable. However, viral replication rebounds, rapidly and precisely, to pre-treatment levels on drug withdrawal in patients [ 52 , 53 , 57 ] and in tissue culture [ 1 ]. This in-vitro data confirm replication is controlled by factors independent of either cellular or humoral immune function. Auto-regulation of HCV replication was confirmed most emphatically in patients undergoing plasmapharesis in whom 60–90% reduction in levels of virus returned to baseline, but not beyond, within 3–6 hours of plasma exchange [ 44 ]. Studies suggesting autoregulation of tobacco mosaic virus replication occurred independent of interferon effects, intrinsic interference or interference by defective virus [ 34 ] confirming this phenomenon is not confined to either animal viruses or cells. These data beg the questions: How does the replicative mechanism "choose" any particular level of replication and how does it return, so accurately, to pre-treatment levels? RNA polymerase control Most cellular enzymes are under some form of kinetic control, usually by product inhibition. While simple negative-feedback product inhibition is sufficient to control enzyme reaction velocity and the rate of product synthesis, it is inadequate to ensure the functional quality of any complex molecules – including proteins – synthesised. The functionality of RNA pol output depends on the functionality of protein(s) translated from any RNA synthesized by RNApol. For viruses, and their polymerase, evolutionary survival – i.e. whether the polymerase, and its viral shell, avoids immune surveillance, gains access to cells, and replicates to infect other hosts – is a function of the properties that the sequence, topological variability and structural integrity of envelope proteins impart. RNA polymerase is responsive to and is influenced by accessory proteins that induce conformational changes to alter both processivity and fidelity [ 20 , 31 ], representing partial "proof of concept" of the mechanism postulated. Evolutionary stability Evolutionary stability requires adaptability to changing environmental circumstances. For viruses, an ability to modulate replication and mutation rates dynamically in response to cellular changes is essential. Viruses intrinsically capable of adaptation to environmental changes, including variations in host density, and evolving cell receptor polymorphisms, immune and other host responses, among other variables, will enjoy a competitive advantage over viruses lacking innate responsiveness. Contrariwise, self-replicating molecules, including viruses, that lack innate adaptability, for whom replication is contingent upon a chance confluence of appropriate cellular conditions – including permissive cell receptors, absence of cell defences and so on – are highly vulnerable to extinction by both adverse environmental changes and competition for scarce intracellular resources by molecules capable of adaptation. For viruses, this adaptability requires antigenic and structural diversity be controlled and, in turn, that means the two critical RNA pol attributes, fidelity and processivity, be dynamically modifiable, and controllable. These linked functional requirements imply a dynamic nexus between the functional output of RNA pol (i.e. envelope proteins) and that polymerase. Homeostatic systems Systems capable of homeostatic regulation (auto-regulation) have the following characteristics: i) an efferent arm that effects changes in response to perturbations of an equilibrium; ii) an afferent arm that measures the systems response to those changes; iii) mechanism(s) by which i) and ii) communicate. The mechanism of viral autoregulation – Replicative Homesostasis – described here requires: i) that viral envelope (Env) proteins interact with viral RNA polymerases (RNA Pol ); ii) that these Env :RNA Pol interactions alter both polymerase processivity and fidelity; iii) that wild-type (consensus sequence) Env wt :RNA Pol complexes cause more rapid, less faithful RNA replication than variant (variant) Env mt :RNA Pol complexes. There is solid evidence for each requirements of replicative homeostasis. The Envelope-Polymerase relationship: Evidence for mechanism A large body of literature, for many viruses, establishes an important relationship between envelope and polymerase proteins and documents that Env proteins influence both RNA Pol processivity and fidelity. First, for HIV, overwhelming evidence suggests HIV polymerases properties, and those of related retroviruses – for example, simian immunodeficiency virus (SIV) and the feline immunodeficiency virus (FIV) – are influenced by Env proteins (for example, [ 9 , 15 , 35 ]. Broadly, these indicate heterologous Env proteins – when administered as live attenuated vaccines [ 71 ], adjuvant enhanced protein vaccine [ 83 ], or as recombinant Env proteins in cell culture [ 64 ] – dramatically alter viral load, and both replication and mutation rates of wild-type virus. Specific examples include data demonstrating HIV Env regions obtained from different patient isolates, when cloned into common HIV-1 backbones, conferred a spectrum of replication kinetics and cytotropisms characteristic of the original Env clone, and independent of either the clones' ability to raise antibody [ 51 ], or the replicative characteristics of the 'native' polymerase backbone [ 51 ]. Similarly, chimeric HIV-1 viruses expressing heterologous Env, again with a common polymerase backbone, have replication kinetics and cell tropism phenotypes identical to the parental Env clone [ 39 ], suggesting the Env is a critical determinant of polymerase function. Similar results obtained with SIV clones [ 36 ] strongly support conclusions drawn from feline immunodeficiency virus [ 37 ] data. Fine mapping of HIV envelope proteins identified 6 mutations within the V1-V3 loop that increased viral replication in a manner independent of nef [ 77 ], confirming other work examining HIV Env recombinants [ 14 ], and extending earlier work that demonstrated a single amino acid substitution (at position 32 of the V3 Env domain) was sufficient to change a low replication phenotype into high-replicating phenotype [ 13 ]. Finally, for HIV, co-transfection with Env variants at 10 fold excess dramatically inhibited replication of wild-type virus [ 75 ], providing direct evidence for both the interaction and differential affinity for wild-type and variant Env for polymerases. Critically, many of these observations are from in-vitro systems, indicating the effects are independent of either cellular or humoral immune influence. Many studies report the effect of Env/polymerase interactions in terms of altered viral tropisms, and did not examine changes to polymerase fidelity explicitly. However, virus replication can alter in only two ways; either there is more or less virus, or the viral genomic sequence may be changed by altered polymerase fidelity. Variant viruses expressing altered envelope proteins will have altered cell receptor affinities and hence, variable cell tropisms. Second, for HCV, many separate observations document HCV replication and polymerase functionality is dependent on envelope proteins: i) HCV viral genotypes are defined by sequences of either envelope or polymerase regions [ 43 , 73 , 74 ] and these are necessarily acquired together – a genetic nexus implying a functional relationship. ii) Observations that a) co-infection with multiple HCV genotypes occurs less frequently than predicted by chance and b) certain HCV genotypes become progressively dominant in populations both suggest – at a population level – replicative suppression of some HCV genotypes by others [ 68 ]. These observations are supported by observations of both homotypic [ 33 ] and heterotypic HCV super-infection [ 32 ] documenting genotype-dependent replicative suppression of one HCV genotype by another in individual patients. iii) Functional infectious chimeric viruses with polymerase and Env proteins derived from different genotypes have not been reported. iv) Full-length HCV chimeras, engineered with deletions of p7 envelope proteins, are replication deficient and non-infections, indicating intact genotype-specific HCV envelope sequences are essential for proper HCV replication. Specific replacement of p7 of the 1a clone with p7 from an infectious genotype 2a clone was replication defective, suggesting a genotype-specific interaction between the p7 envelope protein and other genomic regions [ 66 ]. v) In two independent chimpanzees studies HCV inoculation resulted in persistent infection only in animals developing anti-envelope (E2) antibodies, whereas failure to produce anti-E2 was associated with viral clearance [ 4 , 62 ], intuitively a highly paradoxical result difficult to rationalize unless E2 proteins are important for sustained HCV replication, as we argued previously [ 45 ]. vi) Finally, for HCV, specific motifs within the [polymerase] NS5 region of HCV in chronically infected patients predict response to interferon [ 19 , 67 ] an observation that makes little sense unless interferon interacts directly with NS5 [polymerase] motifs, as in-vitro studies suggest [ 10 ]. Third, HBV envelope and polymerase protein genes have overlapping open reading frames and significant alterations in envelope and polymerase gene and protein sequences cannot, therefore, occur independently, a genetic nexus again implying an important functional relationship. Mutations in envelope sequences occurring spontaneously [ 82 ] following therapy of HBV with lamuvidine and immunoglobulin prophylaxis [ 6 , 72 ] or after vaccine escape [ 8 ] are frequently associated with high level viral replication, although replication-deficient mutations are described [ 47 ]. These data are generally interpreted to mean polymerase gene mutation(s) cause altered polymerase protein sequence and, hence, abnormal polymerase function. While this is probably partially true if the functionally relevant HBV RNA polymerase is an envelope/polymerase heterodimer (analogous to the p66/p51heterodimer of HIV RT [ 30 ]), then an equally valid interpretation is that mutations in envelope genes may change envelope protein conformation and therefore alter normal envelope/polymerase interactions, thus altering processivity and fidelity of the replication complex. This latter interpretation is convincingly supported by data demonstrating that abnormal polymerase function of HBV envelope variants is reversed by co-transfection of Hep G2 cells with clones expressing wild-type envelope sequences [ 81 ] and is further supported by clinical studies demonstrating administration of exogenous HBsAg (protein) to patients with chronic HBV dramatically reduced HBV replication [ 60 ]. Fourth, studies of the coliphage Qβ demonstrate phage coat proteins bind to genomic RNA [ 86 ]to strongly inhibit (association K ic ≈ 10 7–8 M -1 , inhibition K i ≈ 10 9 M -1 s -1 ) [ 79 ] RNA replication by direct suppression of polymerase activity by envelope proteins [ 18 ]. This interaction is dependent on the binding site conformation, but not RNA sequence[ 86 ], suggesting interaction avidity will vary as an inverse function of protein sequence divergence from wild type, an intuitive expectation confirmed experimentally [ 79 ]. An impressive body of literature documents similar relationships between envelope and polymerase function in swine fever, tobacco mosaic [ 34 ], brome mosaic [ 2 ] and other RNA viruses. Importantly, studies of the tobacco mosaic virus confirmed this effect to be host-independent and virus-specific inhibition of viral RNA synthesis and to be quite distinct from any interferon effects, intrinsic interference or interference by defective virus [ 34 ]. Thus, there exists solid evidence for each necessary component of replicative homeostasis for HCV, HBV and HIV, and other viruses. Replicative homeostasis: proposed mechanism Replicative homeosatsis results from differential interactions of wild-type (Wt) and variant (Mt) envelope proteins on RNA pol in a series of feedback epicycles linking RNA pol function, RNA replication and protein synthesis (Figure 4 , 5 ). Intracellular accumulation of variant viral proteins causes progressive, direct, inhibition of RNA pol and also block Env Wt :RNA pol interactions that increase replication and mutation. Progressive blockade of RNA pol by variant envelope results in a less processive, more faithful, polymerase, increasing the relative output of wild-type envelope RNAs, and, subsequently, translation of wild-type envelope proteins and, hence, an inexorable progression to stable equilibria. Quasispecies stability, and other consequences (including immune escape and low-level basal replication), are inevitable outcomes that result from equilibria reached because of these interactions (Figure 5 ). We suggest these interactions, and the resulting equilibria, are important therapeutic targets, and the effective ligands – envelope proteins or topologically homologous molecules – implicit within this hypothesis. Figure 4 Mechanism of replicative homeostasis. At A, relatively high concentrations of Env Wt (blue, A) favour high affinity Env:RNA pol interactions out-competing variant forms (Env mt , red), increasing RNA pol processivity but reduced fidelity increasing relative output of variant RNAs. Subsequent ribosomal (R, mauve) translation increases concentrations Env mt (red), relative to Env Wt , returning the system to equilibrium. Relative excess Env mt (B, red) out-compete Env Wt (blue) for interactions with RNA pol , favouring Env mt :RNA pol , and blocking Env Wt :RNA pol interactions. Env mt :RNA pol complexes relatively decrease RNA pol processivity but increase fidelity, increasing output of wild-type RNAs. Subsequent increased translation of Env Wt relative to Env mt restores the equilibrium. Figure 5 Conseqences of replicative homeostatic cycles. Disturbance to intracellular replicative homeostatic cycles. Events increasing intracellular Env Wt : Env mt ratio (exogenous addition of Env Wt , antibody recognition of Env mt ) will favour Env Wt :RNA pol interactions, increasing RNA pol processivity and reducing fidelity increasing relative output of variant virus. Conversely, events decreasing intracellular Env Wt : Env mt ratios (exogenous addition of Env mt , antibody recognition of Env Wt ) will favour Env mt :RNA pol interactions, decreasing RNA pol processivity and increasing fidelity, thus reducing replication. Viral polymerases are clearly the effector mechanism – the efferent arm – that determines rate of viral RNA replication and mutation. The afferent arm needs to measure both the rate of viral replication and degree of viral mutation. Intracellular envelope concentrations are a direct function of effective viral replication, while competition between wild-type and variant envelope proteins for interaction with RNA pol allows determination of viral mutation rates. Envelope proteins, as opposed to other viral products, are the obvious products to examine for functional variability, and must form part of the afferent arm necessary to "sense" perturbations in the viral equilibrium. While other viral products could be "sensed" to gauge effective viral replication, only functional measurement of envelope protein concentration and topological variability simultaneous measures both the rate of viral replication and envelope functions – properties determined by envelope structure and antigenic diversity – essential for viral survival; immune escape and cell access. Furthermore, envelope and polymerase proteins are typically coded at transcriptionally opposite ends of the viral genome; replication contingent upon a dynamic nexus between envelope and polymerase proteins is, therefore, a functional check of the integrity of the entire viral genome. Importantly, this facet of replicative homeostasis is a direct mechanism of Darwinian selection operating at a molecular level, that ensurs preferential selection and replication of "fit" viral genomes, and maintenance of genotypes (species). Viruses, notably HIV, produce many accessory proteins (such as HIV Nef, gag, rev and HBeAg) that affect viral replication and mutation rate. However, these proteins are encoded within envelope open reading frames (ORFs) or are contiguous with them and are likely to alter functionally with any mutation affecting envelope sequences (Figure 6 ). While these accessory proteins may interact with RNA pol (with or without Env) to reset replicative equilibrium (by changing replication rate or mutation frequency or both), stable equilibria will still result providing the sum effect of variant proteins encoded within the envelope ORF is to decrease RNA pol processivity (v) and mutation (M u ) frequency relative to wild-type protein polymerase interactions. Figure 6 Phenotypic effects of RNA quasispecies complexity. Two-dimensional representation of multi-dimensional hyperdense sequence-space that define viral quasispecies; vast RNA /proteins populations progressively divergent from consensus sequence (0). As genetic the distance of RNAs increases from consensus sequence the amino acid sequence, conformation, and functional properties of resulting proteins may also change, potentially resulting in proteins that, despite originating from identical [consensus sequence] genetic domains, have diametrically opposed function. As many accessory proteins (for example, HIV rev, tat, nef and HCV HP7) have open reading frames contiguous with Envelope, sequence changes to Env will also affect accessory protein function. Testing the hypothesis This hypothesis is simply tested. Manoeuvres that increase intracellular concentrations of variant envelope proteins or decrease wild-type envelope proteins should inhibit viral replication and reduce mutation rates. Conversely, manoeuvres increasing intracellular [Env Wt ] or reducing intracellular [Env mt ] should accelerate viral replication and mutation. In fact, observations relevant to every aspect of this hypothesis have been reported in a variety of systems and circumstances. All outcomes are completely consistent with those predicted by replicative homeostasis. Replicative homeostasis predicts, for example, HCV E2 proteins derived from genotype 1 HCV sequences would reduce HCV replication when administered to patients with heterologous HCV infection (genotypes 2,3 or 4, for example) and studies examining heterologous envelope proteins as direct RNA pol inhibitors are underway. Discussion Replicative homeostasis immediately resolves the paradox RNA viral quasispecies stability and explains how these viruses persist and, thereby, cause disease. Replicative homeostasis also explains the initial decline of viral replication, resolving the kinetic paradox, rationalizing the dynamics of chronic viral infection and other enigmatic and unresolved viral behaviours. Most importantly, replicative homeostasis implies a general approach to antiviral therapy. The equilibria formed by replicative homeostasis are responsive to disturbance of envelope concentrations ensuring viral mutation is neither random nor passive but highly reactive to external influence: Sustained reduction of viral envelope (by immune or other mechanisms) would favour high affinity Env Wt : RNA pol interactions that, in turn, increase polymerase processivity but reduce fidelity accelerating synthesis of variant viral RNAs and, consequently, increased translation of antigenically diverse proteins, reactively driving quasispecies expansion and generating the extreme antigenic diversity of RNA quasispecies. Alternatively, in the absence of immunological recognition, variant envelope / polymerase interactions predominate, restricting viral replication and mutation, thus maintaining basal output of consensus viral sequences, thus maintaining genotype. Immune escape and maximal cell tropism are inevitable consequences of the potential antigenic diversity generated by RNA replication mediated by the reactive equilibria of replicative homeostasis. Potential viral antigenic diversity is numerical superior to any immune response; Theoretically, a small envelope protein of 20 amino acids could assume 20 20 (about 10 26 ) possible conformations, greatly exceeding the ~10 10 antibody [ 80 ] or CTL receptor conformations either humoral and cellular immune responses can generate. A direct consequence of this mismatch and the stable reactive, equilibria resulting from replicative homeostasis is that once infection is established, the clinical outcome is primarily determined by the viruses' ability to maintain control of the quasispecies, rather than the hosts' response to that quasispecies. This sanguine view is supported by both general clinical experience and by kinetic analysis of chronic viral infection (Figure 2 ); if host responses are unable to clear virus at 10 5–7 viral equivalents / ml they are not likely to be any more effective at 10 8–11 eq/ ml. The varied clinical outcomes of viral infections are explained by replicative homeostasis and its failure: Viral failure to down-regulate replication by RNA pol inhibition would cause rapidly progressive or fulminant disease (characterised by massively polyclonal, but ultimately ineffectual, immune responses), while inadequate replication or generation of diversity will result in viral clearance (Figure 3b ). Stable, homeostatic replicative equilibria will result in chronic infection with episodic fluctuations in viral replication and host responses (eg ALT; [ 65 ]) typical of chronic hepatitis or HIV. The widely varied spectrum and tempo of viral diseases, that for viral hepatitis ranges from asymptomatic healthy chronic carriage to fulminant liver disease and death within days, is far more rationally explained on the basis of a broad spectrum of polymerase properties than highly variable and unpredictable (yet genetically homogeneous) immune responses. Homeostatic systems functioning without external perturbations – such as thermostatically controlled water tanks – progress rapidly to stasis (Figure 7 ). In tissue culture, viruses – replicating without immune challenge – are unable (and do not need) to generate antigenic diversity by replicative homeostasis, a phenomenon probably responsible for attenuation of virulence of serially passaged virus cultures. By contrast, in dilute viral culture, where viral envelope and polymerase exist in low concentrations, high affinity Env Wt /polymerase interactions preferentially occur over lower affinity Env mt /polymerase interactions, replicative homeostasis predicts increased viral replication and mutation would occur and this has been confirmed [ 70 ] Figure 7 Homeostatic systems . In absence of external influence, homeostatic systems (A) progress rapidly to stasis (0) while external perturbations (arrows, e.g. immune recognition of virus) cause pseudo-chaotic fluctuating long-term behaviours in complex systems (B). Perturbations of relative intracellular wild-type and variant envelope concentrations alter RNA pol :Env interactions disturbing the replicative equilibria of replicative homeostasis. Antibodies (or CTL) will alter extracellular concentrations of Env proteins, thus changing intracellular envelope concentrations once extracellular /intracellular Env concentrations equilibrate. Therefore, antibodies to heterologous envelope proteins – developing, for example, during immunization against other viruses or heterotypic co-infection – will reduce relative intracellular concentrations of variant envelope, favouring RNA pol :Env Wt interactions, thus enhancing replication and increasing mutation rates, a prediction confirmed in practice [ 38 , 56 ]. Contrariwise, antibodies to wild-type surface proteins – for example, during administration of anti-HBsAb following liver transplantation for HBV [ 63 ] – would reduce viral replication (Figure 6 ), as seen in practice. Disturbance of viral replicative equlibria by heterologous extracellular antibodies rationally explains antibody-dependent enhancement (ADE) of HIV [ 23 ], Dengue [ 26 ], Murray Valley encephalitis[ 84 ], Ebola [ 78 ] Coxsackie [ 24 ] and other viruses. Similarly, increased HIV replication and mutation after influenza [ 38 ] or tetanus [ 56 ] vaccination; reduced HIV replication during measles [ 50 ] and Dengue [ 85 ] co-infection; clearance of HBV without hepatocyte lysis or evidence of T cell dependent cytotoxicity[ 25 ], are also explained by this mechanism. Previously unexplained and problematic viral behaviours and host responses, including long-term non-progression of HIV [ 7 ]; persistence of transcriptionally active HBV despite a robust immune response [ 48 ]; long-term antigenic oscillations [ 54 ]; spontaneous reactivation of HBV[ 41 ] (among many other viruses); and hypermutation of HIV, for example, all rationally resolve within this conceptual framework. There are clear and quite specific therapeutic implications of replicative homeostasis, as well as more general implications. The envelope/polymerase interactions of replicative homeostasis suggested herein are obvious therapeutic targets, and a site of interferon action: Heterologous envelope proteins from different viruses or genotypes of the same virus, or their structural homologues, are likely to inhibit viral replication, as suppression of HIV replication during measles [ 50 ] and Dengue [ 85 ] co-infection suggests. Interferon is ineffective for HIV and many patients with HBV, and its efficacy in HCV is highly genotype-dependent, strongly implying a direct, virus-specific action unrelated to "immune enhancement", as in-vitro data [ 10 ] and clinical kinetic studies imply [ 52 ]. Complexing of interferon to RNA pol to reduce processivity and increase fidelity would explain both the genotype specificity of interferon action and the kinetics of action and, incidentally, the apparent "immune enhancement" [ 59 ] caused by interferon; if interferon reduces RNA pol processivity while increasing its fidelity, viral RNAs synthesized will contain fewer mutations causing synthesis of antigenically restricted proteins, thus presenting a more homogeneous target susceptible to immune attack. Replicative homeostasis may alter perceptions of strategies underpinning the immune responses. It is possible the primary purpose of the initial polymorphic humoral response to viral infection – typically pentameric IgM – is to push viral replication towards equilibria favouring production of homogeneous virus, thus facilitating a concerted and more focussed humoral and/or cytotoxic T cell response; Strong neutralizing IgG antibodies – antiHBsAg, for example – may develop as a consequence of initially restricted viral replication and mutation permitting effective and specific immune recognition, rather than being the proximate cause of it. The temporal profile of HBsAb, that develops well after HBVreplication falls, strongly supports this view. However, once developed, high-affinity neutralizing antibodies against wild-type virus ensure variant envelope proteins remain dominant within cells, thus maximising polymerase inhibition and inhibiting viral replication. Replicative homeostasis is an adaptation that facilitates stable viral replication in cells and maximises probability of cell-to-cell (and host-to-host) transmission, a prerequisite for viral survival on an evolutionary time scale (Figure 3 ). A subtle, more primordial, and evolutionary function of envelope/polymerase interactions may explain the origins of replicative homeostasis; Polymerase function contingent upon recognition of, and response to, complex three-dimensional complementarities between polymerase and envelope proteins constitutes a sophisticated encryption technique, effectively "locking" the polymerase, thereby minimises the likelihood any competing RNA (or DNA) molecules are replicated even if correct 5' transcription initiation sequences are present. This is, again, a powerful mechanism of selection, speciation and genotype preservation. As Spiegleman suggested originally [ 55 ], in the fierce competition for finite intracellular resources, reproductive strategies that maximise proliferation of "self" genes, while thwarting propagation of "rival" genes, are strongly selected for, and are highly conserved in evolution. The interferons, and other cytokines, are cellular defence mechanisms that long antedate the immune system. If the interferons are functionally homologous to viral envelope proteins, and interact with viral RNApol to reduce processivity and replication to restrict viral replication and antigenic diversity, increasing their susceptibility to immune clearance, it is possible these genes were acquired as result of positive selection of beneficial virus-cell symbiosis occurring early in eukaryotic cellular evolution, a process responsible for retention of other genes [ 28 ]. Although proposed specifically to explain RNA viral quasispecies stability, replicative homeostasis is, fundamentally, a mechanism that regulates RNA transcription and modulates protein expression. If proteins (i.e. phenotype) modulate RNA pol properties (in a manner contingent on that proteins functionality) and modulate mutations introduced into the RNA templates RNA pol synthesises, a subtle form of "quality control" is exerted over protein synthesis [ 69 ]. This mechanism accelerates, and directs, adaptation: While introduction of lethal mutations to most RNA genomes may not adversely influence quasispecies, replicative homeostasis ensures any RNA mutations that do arise, and that result in beneficial phenotype(s), will favour replication of that RNA molecule, ensuring that phenotype is retained within the quasispecies. Minor change to polymerase fidelity will profoundly effect a quasispecies; as Haldane demonstrated [ 27 ], a reproductive advantage of only 0.1% is sufficient to increase a gene frequency from 0.1% to 50% over a few thousand generations (~1 year for the average patient with HCV) and this effect, therefore, represents a major moulding force in evolution. Thus, replicative homeostasis provides a powerful counterbalance to Muller's ratchet [ 17 ] and, by promoting retention and transmission of acquired phenotype, is a Lamarkian mechanism fully consistent with Darwinian principles and operative at a molecular level. Finally, accessory proteins that alter the processivity and fidelity of both DNA-dependent RNA polymerases [ 31 ] and DNA-dependent DNA polymerases [ 42 ] to modulate polymerases activity are strongly conserved in evolution, suggesting a critical cellular function. Control of DNA-dependent RNA pol transcription by DNA viruses, cellular micro-organisms (e.g. malaria), and eukaryotic cells, subtly modulating cell-surface protein expression, via replicative homeostasis, to mediate immune escape, control cell division and differentiation, or other functions would not be surprising. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552327.xml |
549713 | Adaptive evolution of centromere proteins in plants and animals | Background Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis . Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif). Results Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection. Conclusions CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi. | Background Centromeres are the chromosomal loci where kinetochores assemble to serve as attachment sites for the spindle microtubules that direct chromosome segregation during mitosis and meiosis. Despite this essential conserved function in all eukaryotes, centromere structure is highly variable, ranging from the simple short centromeres of budding yeast, which have a consensus sequence of approximately 125 base pairs (bp) on each chromosome, to holokinetic centromeres that span the entire length of a chromosome [ 1 ]. In plants and animals, centromeres are large and complex, typically comprising megabase-sized arrays of tandemly repeated satellite sequences that are rapidly evolving [ 2 ] and may differ significantly between closely related species [ 3 - 5 ]. The failure of conventional cloning and sequencing assembly tools to adequately characterize rapidly evolving satellite sequences at centromeres has made them the last regions of most eukaryotic genomes to be well understood [ 1 ]. Although there is no discernable conservation of centromeric DNA sequences in disparate eukaryotes, considerable progress has been made in identifying common proteins that form the kinetochore [ 6 ]. A universal protein component of centromeric chromatin found in all eukaryotes that have been examined is a centromere-specific variant of histone H3 (CenH3), which replaces canonical H3 in centromeric nucleosomes [ 7 , 8 ]. CenH3s are essential kinetochore components yet, like centromeric DNA, they are rapidly evolving [ 1 ]. In both Drosophila [ 9 ] and Arabidopsis [ 10 ], this rapid evolution of CenH3s is associated with positive selection (adaptive evolution), and involves regions of CenH3 that are predicted to contact the centromeric DNA [ 9 , 11 , 12 ]. The finding of positive selection in a protein that is required at every cell division is remarkable. Ancient proteins with conserved function are expected to be under negative selection because they typically have achieved an optimal sequence, so new mutations tend to produce deleterious variants that are quickly eliminated from populations. The canonical histones are extreme examples of this type of protein. In contrast, recurrent positive selection generally occurs as a consequence of genetic conflict, for example in the 'arms race' between pathogen surface antigens and the immune-cell proteins that recognize them. In this case, a mutation in a surface antigen that allows the pathogen to escape detection and proliferate will trigger selection for a new immune receptor to fight the mutated pathogen, which can then mutate again, and so on. The evidence for positive selection of CenH3 proteins specifically in the regions that contact DNA thus suggests a conflict between centromeric DNA and a histone component of the nucleosome that packages it. Is it commonplace for eukaryotes to have such a conflict at their centromeres? Is the conflict unique to centromere-specific histones, or are other proteins that bind centromeres also involved in this conflict? Is conflict responsible for centromere complexity? To answer these questions, we investigated the evolution of a second common DNA-binding kinetochore protein. Of the handful of essential kinetochore proteins that are widely distributed among eukaryotes, only one class other than CenH3 has been shown to bind centromeric DNA: centromere protein C (CENP-C), a conserved component of the inner kinetochore in vertebrates [ 13 - 16 ]. Human CENP-C binds DNA non-specifically in vitro [ 17 - 19 ] and binds centromeric alpha satellite DNA in vivo [ 20 , 21 ]. Vertebrate CENP-C and the yeast centromere protein Mif2p [ 22 , 23 ] share a 24 amino-acid motif (CENPC motif) that has also been found in kinetochore proteins in nematodes [ 24 ] and plants [ 25 ]. As expected for kinetochore proteins, disruption or inactivation of genes encoding proteins containing a CENPC motif (CENP-Cs) results in the failure of proper chromosome segregation [ 16 , 23 , 24 , 26 - 28 ]. Other than the defining CENPC motif, these proteins are dissimilar in sequence across disparate phyla. Such a small stretch of sequence conservation, accounting for less than 5% of the length of these 549-943 amino-acid proteins, is unexpected considering that CENP-Cs are encoded by essential single-copy genes that are expected to be subject to strong negative selection. We therefore wondered whether the same evolutionary forces responsible for the rapid evolution of CenH3s cause divergence of CENP-Cs outside of the CENPC motif. Here, we describe coding sequences from several unreported Cenpc genes and test whether Cenpc genes are in general, like CenH3 genes, subject to positive selection. We find evidence for adaptive evolution of CENP-C in plants and animals, but we find negative selection in yeasts. Our results provide support for a meiotic drive model of centromere evolution. Results and discussion CenH3s evolve under negative selection in some lineages Previous work has shown that CenH3s are evolving adaptively in Drosophila and Arabidopsis [ 9 , 10 ], but their mode of evolution in mammals is not known. Selective forces acting on proteins can be measured by comparing the estimated rates of nonsynonymous nucleotide substitution (K a ) and synonymous substitution (K s ) between coding sequences from closely related species. These rates are expected to be equal if the coding sequences are evolving neutrally (K a /K s = 1). Negative selection is indicated by K a /K s < 1, and positive selection is indicated by K a /K s > 1. To obtain a pair of closely related mammalian CenH3s, we used the sequence of the mouse ( Mus musculus ) CenH3, CENP-A [ 29 ], to query the High Throughput Genomic Sequences portion of the GenBank database [ 30 ] with a tblastn search, and identified a rat ( Rattus norvegicus ) genomic clone (AC110465) that contains the predicted rat CENP-A coding sequence. The predicted CENP-A protein is encoded in four exons and is 87% identical in amino-acid sequence to mouse CENP-A, excluding a 25 amino-acid insertion that appears to derive from a duplication of the amino terminus (Figure 1 ). This gene model is partially supported by an expressed sequence tag (EST; BF561223) that includes the first three exons, but which terminates in the predicted intron 3. To determine whether Cenpa is evolving adaptively in rodents, we compared K a and K s between mouse and rat using K-estimator [ 31 ]. Positive selection in single-copy genes that are essential in every cell is expected to be localized and more difficult to detect than in nonessential genes or members of multigene families because of simultaneous negative selection to maintain their essential functions. In Drosophila and Arabidopsis , CenH3s are under positive selection in their tails, but also under negative selection in much of their histone-fold domains. We therefore used the sliding-window function of K-estimator to scan through the coding sequences using 99 bp windows every 33 bp in an effort to find regions of positive selection. This analysis detected statistically significant negative selection for all of the windows except one that failed to rule out neutrality, indicating that CENP-A is under negative selection (K a = 0.11, K s = 0.33; K a < K s with p < 0.001) in both the tail and the histone-fold domains. Similar results were obtained when comparing either sequence with the Cenpa gene from Chinese hamster ( Cricetulus griseus ) [ 32 ], although the greater divergence (K s = 0.45 rat, 0.67 mouse) makes the statistical conclusion near the limit of reliability (K s ≤ ~0.5) because of the increased likelihood of multiple substitutions. Thus, CENP-A appears to have been under negative selection throughout its length in multiple rodent lineages. We also compared the human Cenpa gene [ 33 ] with the Cenpa gene from chimpanzee ( Pan troglodytes ). A blastn search of the Genome Sequencing Center's assembly of the chimpanzee genome [ 34 ] using human Cenpa identified the chimp Cenpa gene encoded in four exons in Contig 286.218. We searched the NCBI trace archives [ 35 ] to verify the sequence and the existence of appropriate putative intron splice sites. The predicted chimpanzee Cenpa gene differs from the human gene by six synonymous nucleotide substitutions and an indel (insertion or deletion) of two codons. This excess of synonymous substitutions indicates negative selection of CENP-A ( p < 0.01). Overall negative selection of CENP-A appears also to extend to the bovine (CB455530) protein, given the relatively high degree of conservation seen for all regions, including the tail and Loop 1 regions that evolve adaptively in Drosophila (Figure 1a ). We also found overall negative selection in CenH3s of grasses. We used the CENH3 gene (AF519807) of maize ( Zea mays ) [ 36 ] to search ESTs [ 37 ] from sugarcane ( Saccharum officinarum ), and identified three that encode full-length CENH3 genes (CA119873, CA127217, and CA142604). The CenH3 proteins encoded by these ESTs differ from each other by 2-4 amino acids. Because sugarcane is thought to be octaploid, these variants may represent co-expressed homeologs. The coding regions of ESTs CA119873 and CA127217 differ by four synonymous and four nonsynonymous substitutions (K s = 0.03, K a = 0.01), suggesting negative selection. Comparison of either of these sequences with maize CENH3 by sliding-window analysis found that all windows had K s > K a , with overall negative selection (K s = 0.24, K a = 0.13; p < 0.01). Thus, in contrast to CenH3s in Arabidopsis and Drosophila , CenH3s of rodents, primates, and grasses appear not to be evolving adaptively. The evident lack of positive selection on CenH3 in mammals and grasses raises the possibility that another kinetochore protein is evolving in conflict with centromeric DNA in these organisms, in which centromeric satellite sequences are known to be evolving rapidly [ 2 , 38 ]. We focused on CENP-C, which is found to co-localize with CenH3 to the inner kinetochore in humans [ 13 ] and maize [ 36 ]. Mammalian CENP-C is evolving adaptively To address the possibility that CENP-C is adaptively evolving in mammals, we used the mouse sequence [ 14 ] as a query in a tblastn search to identify Cenpc ESTs from rat. From these ESTs (see Additional data file 1), we obtained and sequenced a full-length cDNA (see Additional data file 2), and compared its coding sequence with that of the mouse Cenpc gene (68% predicted amino-acid identity). We found positive selection over most of the amino-terminal two-thirds of the coding sequence, interrupted by one region of significant negative selection (mouse codons 208-273), one region of nearly significant negative selection (mouse 410-464), and three short regions without significant selection (Figure 2a ; Table 1 ). Most of the carboxy-terminal one-third of the protein, including the CENPC motif and an additional region that is homologous to the budding yeast CENP-C protein Mif2p [ 22 , 23 ], has been under negative selection. We conclude that at least some regions of Cenpc genes are evolving adaptively in rodents. To determine whether any of these regions is also under positive selection in primates, we identified the Cenpc gene of chimpanzee by using the human Cenpc coding sequence (GenBank accession number M95724) to search the assembled chimpanzee genome and the NCBI trace archives. We found that the chimpanzee genome contains a single copy of the Cenpc structural gene (contigs 375.88-375.100), as well as a processed Cenpc pseudogene (contigs 76.642-76.643), as has been found in humans [ 14 , 18 , 39 ]. The predicted chimpanzee Cenpc coding sequence differs by 17 nucleotide substitutions from the human cDNA sequence, with K s = 0.0054 and K a = 0.0063. The > 99% identity of the human and chimp coding sequences provides little opportunity to detect selection, but using sliding-window analysis we found a single region of significant positive selection (human codons 278-585) that overlaps the central regions of positive selection found in the more divergent rat-mouse comparison, indicating that the central portion of CENP-C is under positive selection in both rodents and primates. To confirm these results, we applied the codeml program of PAML [ 40 ] to a multiple sequence alignment of mammalian CENP-Cs. PAML calculates the likelihood of models for neutral and adaptive evolution based on a tree and estimates K a /K s ratios. We compared the null model with two fixed site classes (K a /K s = 0 or 1) to a 'data-driven' model in which two classes of sites were estimated from the data. The data-driven model was found to be significantly more probable than the null model (χ 2 = 8.7; p = 0.01) with K a /K s = 0.20 for 57% of the 685 sites in the multiple alignment and K a /K s = 1.64 for 43% of the sites (data not shown). Similar results were obtained using either a DNA- or a protein-based tree, or testing more complex models. When the same tests were applied to the core region of 11 aligned Brassicaceae (mustard family) CenH3s, only 17% of residues were estimated to be in the positive selection class (K a /K s = 2.54) ([ 11 ] and data not shown), which indicates that positive selection on mammalian CENP-C has occurred more extensively than on CenH3s. Amino-acid sites of positive selection in mammalian CENP-Cs were identified as those with significant posterior probabilities. These were found to be scattered throughout the multiply aligned region with 5 of the 18 highly significant sites prominently clustered within 25 residues (human codons 424-448) in a region of positive selection identified by K-estimator analysis. Therefore, pairwise K-estimator and multiple PAML analyses yield similar results and reveal that large regions of mammalian CENP-Cs have been adaptively evolving. Adaptively evolving regions overlap DNA-binding and centromere-targeting regions The regions of positive selection in rodent and primate CENP-Cs overlap some protein landmarks identified in functional analyses of human CENP-C. The binding activity of human CENP-C to DNA in vitro has been mapped by two groups of investigators. Sugimoto and colleagues [ 17 , 18 ] found that the region including amino acids 396-498 bound DNA and was stabilized by including flanking amino acids on one or both sides (330-498 or 396-581; Figure 3a ), suggesting that at least two regions in the central portion of the protein contribute to DNA binding. Yang and colleagues [ 19 ] identified two non-overlapping DNA-binding regions: amino acids 23-440 and 459-943. They found a weak DNA-binding activity at the carboxyl terminus in region 638-943, which includes the CENPC motif (737-759) and the conserved Mif2p-homologous region (890-941). This suggests that region 459-943 itself contains at least two DNA-binding regions, a weak one at region 638-943, and a stronger one that may correspond to region 396-581 described by Sugimoto and colleagues. Both the central region and the carboxyl terminus have been shown to bind DNA in vivo [ 21 ]. Comparison of the regions of positive selection found in rodents and primates with these DNA-binding regions reveals extensive overlap with the central DNA-binding regions (Figure 3a ), including the cluster of highly significant sites between codons 424 and 448 identified by PAML analysis. This is consistent with previous evidence that adaptive evolution of CenH3s occurs in regions that have been implicated in DNA binding [ 9 , 11 ]. No positive selection was observed for the poorly mapped carboxy-terminal DNA-binding domain in our sliding-window analysis, suggesting either that this DNA-binding domain is not evolving adaptively or that strong negative selection on the CENPC motif can obscure detection by our sliding-window analysis of positive selection on nearby amino acids that contact centromeric DNA. In the DNA-binding Loop 1 region of Arabidopsis CenH3, adaptively evolving codons are found in close proximity to codons under strong negative selection [ 11 ]. In human CENP-C, three regions have been reported to confer centromere targeting. One targeting signal was recently reported in region 283-429 [ 41 ]. A second targeting region was mapped by mutation to region 522-534, with arginine 522 crucial for localization [ 42 ]. Targeting by the conserved carboxyl terminus (728-943) occurs for species as distant as Xenopus [ 21 , 41 - 43 ]. A segment that includes both the first and second targeting regions (1-584) failed to confer targeteting to centromeres in hamster BHK cells, however [ 43 ]. We find that these two targeting regions are within the region of positive selection in primates and overlap with three of the regions of positive selection in rodents. A correspondence between centromere targeting and adaptive evolution has been noted for Drosophila CenH3, where the adaptively evolving Loop 1 region has been shown to be necessary and sufficient for targeting when swapped between native and heterologous orthologs [ 44 ]. Therefore, the lack of centromeric targeting of a human CENP-C fragment containing the first and second targeting regions in the heterologous hamster system might be attributed to adaptive evolution of DNA-binding specificity in these regions. Targeting of native CENP-C proteins depends on other centromere proteins that vary according to species [ 45 ], but the dependence of CENP-Cs on CenH3s for targeting appears to be universal [ 24 , 46 - 49 ]. This dependence suggests that CENP-C proteins contain a conserved CenH3-interacting region, for which the CENPC motif is the only obvious candidate. The first half of the CENPC motif is rich in arginines, whereas the second half has mixed chemical properties including three aromatic residues (Figure 3c ). In the non-specific binding of nucleosome cores to DNA, 14 DNA contacts are made by arginines binding to the minor groove [ 50 ]. This suggests that the weak DNA binding of the carboxyl terminus of CENP-C may be mediated by the arginines of the CENPC motif, with the remainder of the motif contacting a conserved structural feature of centromeric nucleosomes. Not all regions of CENP-C that display positive selection correspond to regions that bind DNA in vitro or that are sufficient for targeting centromeres. For example, the region comprising the most amino-terminal 200 or so amino acids of rodent CENP-C has been evolving adaptively, but the orthologous region in human CENP-C fails to bind DNA in a southwestern assay [ 17 , 19 ] or to localize to centromeres of human embryonic kidney cells [ 21 ]. This suggests that the amino-terminal region of CENP-C plays a supporting role in packaging centromeric chromatin. A parallel situation appears to hold for the adaptively evolving amino-terminal tail of Drosophila CenH3, which was found to be neither necessary nor sufficient for targeting in vivo to homologous centromeres. In this case, Loop 1 was identified as the targeting domain, and the amino-terminal tail was hypothesized to help stabilize higher-order chromatin structure by binding to linker DNA, similar to the known binding activity of canonical histone tails [ 44 ]. If CENP-C in mammals is subject to the same evolutionary forces that shape the adaptive evolution of the CenH3 tail in Drosophila , then CENP-C might be playing a comparable role in the stabilization of higher-order centromeric chromatin. Positive selection in the central DNA-binding and centromere-targeting region of CENP-C offers an explanation for the lack of conservation of this region between chicken and mammals [ 51 ]: as positive selection acts on the amino acids that contact rapidly evolving centromeric satellites and that serve to target the protein to a specific but ever-changing substrate, it may eventually erase all recognizable homology in these protein regions. Cenpc gene structure and conservation in plants Our finding that adaptive evolution is occurring in animal CENP-Cs encouraged a similar survey of plant CENP-Cs, because centromeres from both animals and seed plants comprise rapidly evolving satellite sequences. At the time we began this study, Cenpc genes in plants had been characterized only in maize ( Z. mays ), so we needed first to identify Cenpc homologs from other plants to ascertain whether or not the gene is evolving adaptively. Three Cenpc homologs have been described in maize: CenpcA, CenpcB, and CenpcC [ 25 ]. Immunological localization of CENP-CA to maize centromeres indicates that it is probably functional, so plant relatives of maize CENP-CA should also represent CENP-Cs. We used the CENP-CA protein sequence (AAD39434) as a query in a tblastn search of GenBank, and identified a single Cenpc homolog (AC013453, At1g15660) in the genome of Arabidopsis thaliana by sequence similarity at both protein termini (Figure 4 ). Isolation and sequencing of a full-length Cenpc cDNA (Additional data file 2) revealed that the 705 amino-acid CENP-C protein of Arabidopsis is encoded in 11 exons, with the CENPC motif encoded in exon 10 (Figure 5 ). Recently, Arabidopsis CENP-C has been found to localize to Arabidopsis centromeres [ 52 ]. We searched the GenBank EST database, querying with the predicted protein sequences of maize CENP-CA and Arabidopsis CENP-C. We identified ESTs from putative plant Cenpc genes in 20 angiosperm species representing eight families and in the moss Physcomitrella patens (see Additional data file 1). We obtained the cDNA clones corresponding to 16 of these ESTs and sequenced them completely (see Additional data file 2). An alignment of the carboxyl termini encoded by cDNAs representing six angiosperm families revealed that the final 80 or so amino acids of CENP-C, including the CENPC motif, are highly conserved in plants (Figure 4b ). For comparison, the carboxyl termini of vertebrate CENP-C proteins have approximately 180 amino acids following the CENPC motif (Figure 3a ), including a block of 52 amino acids that is conserved in yeast Mif2p [ 22 , 23 ], but not in nematodes [ 24 ]. The carboxyl termini of plant CENP-Cs do not show significant similarity to animal and fungal CENP-Cs except for the CENPC motif. As an aid in identifying other conserved regions of angiosperm CENP-Cs, we developed gene models for full-length Cenpc cDNAs by aligning them with available genomic sequences (Additional data file 1). A full-length cDNA from barrel medic ( Medicago truncatula ) encodes a protein of 697 amino acids, which corresponds to a gene model of eleven exons when aligned to a genomic pseudogene (Figure 5 ). We also predicted gene models for Cenpc genes in the grasses using cDNAs and genomic sequences from rice ( Oryza sativa ), maize, and sorghum ( Sorghum bicolor ) (Figure 5 ). The maize gene model of 14 exons suggests an explanation for the anomalous maize cDNA ' CenpcC ' (AF129859) [ 25 ], which differs from all other plant Cenpc s in encoding an unrelated carboxyl terminus. CenpcC is 99.9% identical to maize CenpcA until it diverges downstream of the CENPC motif at the point corresponding to the end of exon 13 in our gene model. On the basis of an overlap with maize and Sorghum genomic sequence that spans the intron between exons 13 and 14, we conclude that the divergent 3' end of CenpcC derives from the unspliced intron 13 of CenpcA , and that all angiosperm CENP-Cs share a highly conserved carboxyl terminus. Comparing the gene models of Arabidopsis , barrel medic, maize, Sorghum , and rice, the limited conservation of the encoded amino-acid sequences and approximate correspondence of exon sizes suggest that the exons in the amino-terminal half and the final two exons of plant CENP-C are conserved (Figures 3 , 5 ). The middle region does not show conservation of intron position or encoded peptide sequence, indicating rapid evolution within angiosperms. We assumed conservation of the first five intron positions in the 5' half of the coding sequence to generate an amino-terminal alignment that represents five families, including the protein encoded by a beet ( Beta vulgaris ) cDNA that appears to contain an unspliced intron. Our alignment reveals short regions of conservation throughout the amino terminus, as well as a high relative incidence of the dipeptide SQ in the poorly conserved exon 5 (Figure 4 ). Despite these short regions of conservation within angiosperms, no sequence similarity between plant and animal CENP-Cs could be detected outside of the CENPC motif. Nevertheless, plant and animal CENP-Cs appear to share an overall architecture (Figure 3 ). Both angiosperm and vertebrate CENP-Cs [ 16 ] have regions of conservation at the amino and carboxyl termini, with little or no conservation in the middle region of the protein. Remarkably, plant and animal CENP-Cs also share the same modular exon organization for the CENPC motif, which lies within a 105-108 bp exon (encoding 35-36 amino acids) that is spliced in the same frame in both plants and animals (see Additional data file 3). Considering the similar overall lengths of plant and animal CENP-Cs, the arrangement of conserved regions, and the common location of the CENPC module, it appears that corresponding regions of the protein are evolving similarly and may serve similar functions. Recurrent exon duplications in the grasses Multiple alignment of plant Cenpc s revealed that one region of the gene is subject to duplication, but only in grasses. One part of the poorly conserved middle region of the gene has been repeatedly duplicated and deleted, thus encoding proteins of different sizes. In rice, an ancestral pair of exons, corresponding to exons 9 and 10 in maize CenpcA , has been triplicated in tandem (Figure 5 ). To facilitate comparison with maize and other grasses, we designated the rice exons as 9a-10a, 9b-10b, and 9c-10c. Exon 9c has an additional internal tandem duplication of its first 14 codons. Consensus sequences derived from overlapping truncated ESTs (Additional data file 1) and cDNAs (Additional data file 2) from the closely related species wheat ( Triticum aestivum ) and barley ( Hordeum vulgare ) indicate that there are two tandem copies of exons 9 and 10 in these species (designated 9p-10p and 9q-10q in Figure 5 ). We confirmed the sequence of these exons by designing primers and amplifying the corresponding regions from wheat and barley genomic DNAs. Single copies of exons 9 and 10 were found in full-length cDNAs from sugarcane, Sorghum bicolor and Sorghum propinquum (Table 2 ; Figure 5 ). Exon duplications were also found for Sorghum species but, surprisingly, these involved a different pair of exons, 11 and 12. One full-length cDNA from S. bicolor has only a single copy of exons 11 and 12, whereas a truncated pseudogene from S. bicolor and a full-length cDNA from S. propinquum are duplicated for exons 11 and 12 (designated 11a-12a and 11b-12b). The S. bicolor pseudogene has a deletion that joins sequences just upstream of the initiation codon in exon 1 to sequences upstream of exon 2. Despite the presence of tandemly duplicated exons, the S. bicolor truncated pseudogene is more closely related to the full-length S. bicolor gene than it is to the S. propinquum gene. Exons 11 and 12 in the S. bicolor full-length gene are identical to 11b-12b in the pseudogene, but have 7 differences from 11a-12a. This suggests that the duplication of exons 11 and 12 preceded the divergence of S. propinquum and S. bicolor , and that the full-length S. bicolor gene may have been derived by loss of exons 11a-12a from a full-length ancestral gene similar to the truncated pseudogene. We wondered why two different pairs of exons, 9-10 and 11-12, were each independently subject to duplication in the grasses. When we examined multiple alignments of the peptide sequences encoded by both exon pairs in Logos format, it became apparent that they resembled each other in length and composition (Figure 6a ). Exons 9 and 11 both encode peptides of 25-28 residues that are rich in acidic amino acids, whereas exons 10 and 12 encode peptides of 30-38 residues that are rich in basic amino acids. We compared alignments of exons 9 and 11 and alignments of exons 10 and 12 using the Local Alignment of Multiple Alignments (LAMA) program, and found that these exon pairs appear to be homologous ( E < 0.0001 for both comparisons). We conclude that exon pairs 9-10 and 11-12 derive from a more ancient duplication event. To trace the likely ancestry of these duplication events, we used an alignment of the exons from multiple species to construct phylogenetic trees of duplicates of exons 9-10 and 11-12 (Figure 6b ). This phylogeny suggests that there have been numerous duplication events in the history of the grasses (Figure 6c and data not shown): first, a duplication generating exons 9-10 and 11-12 in an ancestor of the grasses; second, a duplication generating exons 9p-10p and 9q-10q; third, a duplication generating exons 11a-12a and 11b-12b in the Sorghum lineage; fourth, two duplications generating rice exons 9a-10a, 9b-10b, and 9c-10c all within the rice 9q-10q lineage; and fifth, a partial duplication in rice exon 9c. There also appear to have been at least three losses of duplications: one of exons 11a-12a in the lineage leading to the full-length S. bicolor gene, one of exons 11b-12b in the sugarcane genes, and one of the hypothetical rice 9p-10p. Alternatively, it is possible that the latter loss and one of the rice-specific duplications resulted from gene conversion of rice 9p-10p by a derivative of rice 9q-10q. Regardless of the exact number of duplication and deletion events, it is clear that the exon pair ancestral to grass exons 9-10 and 11-12 has been subjected to repeated episodes of duplication and deletion. Plant CENP-Cs are adaptively evolving The delineation of gene models for plant Cenpc s allowed us to analyze them for evidence of adaptive evolution. First, we compared Cenpc s from Arabidopsis species in which we had previously found adaptively evolving CenH3s. Using the A. thaliana genomic sequence to design primers, we amplified, cloned, and sequenced a Cenpc cDNA from A. arenosa (Additional data file 2). Comparing this sequence with that of A. thaliana, the predicted proteins differ by 87 amino-acid subtitutions out of 703 alignable residues, plus five indels of 1-3 amino acids. We applied the sliding window option of K-estimator to the aligned coding sequences of A. thaliana and A. arenosa Cenpc . At three regions, K a exceeded its 99% confidence interval for the null hypothesis, indicating that these regions are under positive selection (Figures 2b , 3 ). These regions correspond approximately to exon 5 (codons 178-221 in the A. thaliana sequence), the 3' half of exon 6 (codons 376-441), and exons 8 and 9 (codons 486-618). In addition, a region encompassing most of exons 1 and 2 (codons 24-89) was found to be under positive selection with p < 0.03. We also determined that the 5' half of exon 6 (codons 255-386) and the conserved exons 10 and 11 (codons 595-703) are under negative selection with p < 0.01. Curiously, an indel at the beginning of exon 9, where the A. arenosa cDNA has a CAG (glutamine) codon that is absent in the A. thaliana cDNA, appears to be caused by the species-specific use of alternative acceptor splice sites, because the genomic sequence (data not shown) at this intron-exon boundary is identical in both species (...cag cag ^GAG GGT... or ...cag ^CAG GAG GGT...). The presence of species-specific alternative splicing of the same codon in an adaptively evolving region suggests that splicing variation can contribute to adaptive variability. To examine whether positive selection in Cenpc is unique to Arabidopsis or occurs more generally in plants, we compared Cenpc genes from the two Sorghum species. We removed the duplicate exons 11a and 12a from the S. propinquum coding sequence in order to compare the sequence with the full-length gene from S. bicolor . For this comparison, K a = 0.014 and K s = 0.003. K a exceeds the 99% confidence interval of the null hypothesis, and neutral evolution can be rejected in favor of positive selection. The limited divergence between these two sequences did not allow statistically significant conclusions to be drawn about positive selection in particular regions of the gene. To address which regions are under positive selection, we compared the S. bicolor sequence with the maize CenpcA coding sequence (75% amino-acid identity). Between maize CenpcA and S. bicolor, K a = 0.12, K s = 0.14, and there are seven indels of 1-11 codons. We identified positive selection for a single window in exon 1, for a region including all of exon 5, for a region in the second half of exon 6, and for a region from the end of exon 12 through most of exon 14 (Table 2 and Figure 2c ). Negative selection was found for a region from exons 1-4, a region in the middle one-third of exon 6, and a region from the end of exon 6 through exon 12 (Table 2 and Figure 2c ). The regions of positive selection seen in exons 1 and 5 clearly overlap the corresponding regions in Arabidopsis (Figure 3b ). Although the region of positive selection seen in exon 6 of the grasses cannot be aligned with that in exon 6 of Arabidopsis because of sequence divergence, they occur in the same general area of the protein. The region of positive selection in exons 12-14 was somewhat surprising given the strong conservation around the CENPC motif, and we wondered if this selection was specific to the maize or Sorghum lineage. To test this possibility, we compared maize CenpcA and S. bicolor Cenpc with a Cenpc gene from sugarcane. Of three sugarcane cDNAs that we obtained (Additional data file 2), two had identical coding sequences ( Cenpc1 ), and the third ( Cenpc2 ) differed by 13 nucleotide substitutions, suggesting that Cenpc1 and Cenpc2 may be homeologous genes in the polyploid sugarcane genome. We compared Cenpc1 to maize CenpcA and S. bicolor Cenpc . Regions of positive or negative selection identified in the maize/ Sorghum comparison were generally found to coincide with regions under selection in the corresponding direction in maize/sugarcane and Sorghum /sugarcane comparisons, although in a few cases the selection was not found at the p < 0.05 level of significance in all comparisons (Table 2 ). We conclude that these regions are subject to recurrent adaptive evolution. In two cases, a region was found to be under significant selection in opposite directions in different comparisons. First, a region in exon 6 (maize codons 232-286) that was not under significant selection in the maize/ S. bicolor comparison was under negative selection in the maize/sugarcane comparison, but under positive selection in the S. bicolor /sugarcane comparison; this suggests that positive selection in S. bicolor and negative selection in maize combined to give a non-significant result in the maize/ S. bicolor comparison. Second, the region of positive selection in exons 12-14 identified from the maize/ Sorghum comparison was under positive selection in the maize/sugarcane comparison, but under negative selection in the Sorghum /sugarcane comparison, indicating that the positive selection in this region is unique to the maize CenpcA lineage (Table 2 , Figure 3b ). Therefore, in some regions of CENP-C adaptive evolution appears to be episodic, as has been seen previously for primate lysozymes [ 53 ]. Phylogeny-based PAML analysis confirms that plant CENP-Cs are adaptively evolving and in an episodic fashion, consistent with inferences based on pairwise K-estimator analysis. As was found for mammalian CENP-C, the data-driven model for grass CENP-C was found to be significantly more probable than the null model (χ 2 = 12.0; p = 0.003) with K a /K s = 0.00 for 51% of the 686 sites in the multiple alignment and K a /K s = 2.00 for 49% of the sites (data not shown). Using the PAML 'free-ratio' option that measures K a /K s differences between branches in a tree [ 54 ], we found that CENP-C is adaptively evolving (χ 2 = 10.0; p = 0.007 for the data-driven over the null model) along both Sorghum lineages (K a /K s = 2.6) and along the sugarcane Cenpc2 lineage (K a /K s = 1.3) but not detectably along the sugarcane Cenpc1 lineage (K a /K s = 0.23). PAML analysis also confirmed that the carboxy-terminal region of maize CenpcA is adaptively evolving (χ 2 = 7.8; p = 0.02 for the data-driven model over the null model) with K a /K s = 1.4. Thus, different methods of analysis demonstrate that CENP-Cs are adaptively evolving in an episodic fashion in grasses that have multiple CENP-C copies. Maize CenpcA is co-expressed with another Cenpc gene, CenpcB [ 25 ], for which incomplete sequence information is available (AF129858, AW062057, and AY109432). The available CenpcB sequence begins in exon 5 and continues through exon 14, and has seven in-frame indels relative to CenpcA . We found negative selection in a region from the end of exon 5 through the first half of exon 6 ( CenpcA codons 205-358, p < 0.01), and positive selection from the end of exon 6 through exon 7 (codons 403-492, p < 0.01). Elsewhere the null hypothesis could not be rejected. Comparing CenpcB with S. bicolor , K a = 0.13 and K s = 0.11, and there are six in-frame indels between the sequences. We found positive selection in the first half of exon 6 ( Sorghum codons 273-327, p < 0.03) and negative selection from exon 10 through the first few codons of exon 13 (codons 537-619, p < 0.02). Elsewhere the null hypothesis could not be rejected. In summary, regions under negative selection in other grass Cenpc s can be under positive selection in CenpcB , and regions under positive selection in other grass Cenpc s are under negative selection or evolving neutrally in CenpcB (Figure 3b ), suggesting that CENP-CB has been subjected to different selective forces since its divergence from CENP-CA. Just as gene duplication can result in different selective pressures on the two genes, duplications within a gene can lead to specialization and thus can change selective pressures on the region. Such specialization appears to have occurred between the anciently duplicated region encoded by exons 9-10 and 11-12 (Figure 6a ). In maize, sugarcane, and Sorghum we detected negative selection for exons 9-12, but in the more recent duplication of exons 9 and 10 in wheat and barley we detected positive selection in a region from the last codon of the first copy of exon 10 to the first four codons of exon 12 ( p < 0.01). Additional windows in exons 9p-10p and 12 had K a > K s ( p > 0.05), suggesting that most of the duplicated region has been evolving adaptively (Figure 2d ). In contrast, in the adjacent conserved carboxy-terminal region (corresponding to CenpcA codons 625-690), we detected only negative selection ( p = 0.01), as though exon duplication allowed for adaptation. We find an approximate correspondence between adaptively evolving regions in angiosperms and those in mammals that overlap DNA-binding and centromere-targeting regions. Although no DNA-binding regions have been experimentally determined for plant CENP-Cs, the correspondence with animal CENP-Cs suggests that the different regions play comparable roles. One of the corresponding adaptive regions is repeatedly duplicated in grasses, and the distribution of basic residues in exons 10 and 12 suggests that the repeat unit binds DNA. A parallel situation again appears to be found for the amino-terminal tails of some Drosophila CenH3s, which contain repeats of a minor groove-binding motif that are thought to provide DNA compositional preference [ 12 ]. Thus, both plant and animal CENP-Cs show adaptively evolving features that parallel those found in CenH3s. Yeast MIF2 is under negative selection If positive selection for CENP-Cs in plants and animals is related to centromere complexity, then we would expect conventional negative selection to operate in organisms such as budding yeast, which have simple centromeres. The MIF2 genes of Saccharomyces cerevisia e [ 26 ] and S. paradoxu s [ 55 ] are 93% identical in amino-acid sequence, with K a = 0.036 and K s = 0.38. In sharp contrast to all pairwise comparisons of plant and animal Cenpc genes, K a was much less than K s for all of the 99 bp windows of yeast MIF2 , indicating that it is under negative selection throughout its length ( p < 0.001). In all pairwise comparisons among these two species and the additional species S. mikatae and S. bayanus [ 55 ], we consistently found evidence of negative selection with K a << K s (range of K a , 0.036-0.093; range of K s , 0.38-0.82). We also found strong negative selection for all 99 bp windows in pairwise comparisons of yeast CenH3 (Cse4p; data not shown). Thus, adaptive evolution of both CenH3s and CENP-Cs appears to be limited to organisms with complex centromeres. Meiotic drive model of centromere evolution We have demonstrated that CENP-C has been adaptively evolving in multiple lineages of both plants and animals, a feature that had been previously shown for some CenH3s. Thus, the occurrence of adaptive evolution appears to be a general feature of proteins that bind to complex centromeres. Recurrent adaptive evolution implies an arms race, and an arms race involving centromeric DNA-binding proteins is remarkable given that centromeres have a conserved function. But centromeric DNA is rapidly evolving in plants and animals, so adaptation of the major centromere DNA-binding proteins would maintain an interface with the conserved kinetochore machinery. Indeed, regions of CENP-C that show evidence of positive selection include DNA-binding and specificity regions, in parallel with previous findings for Drosophila and Arabidopsis CenH3 [ 9 , 11 , 44 ]. A 'meiotic drive' model has been proposed to explain the rapid evolution of centromeric DNAs and CenH3s [ 1 ]. According to this model, centromeres compete during female meiosis for inclusion in the single meiotic product that becomes the egg nucleus and so gets transmitted to the next generation. In both animals and seed plants, which of the four meiotic products becomes the egg nucleus is determined by its position in the female tetrad. A centromere variant will increase in the population if it achieves an orientation in female meiosis resulting in its inclusion in the egg nucleus more frequently than its competitors. For example, an expansion of a satellite array may lead to a 'stronger' centromere variant with an expanded kinetochore that attracts more microtubules and results in a slightly greater probability of a favorable orientation in female meiosis. The mechanism of such orientation is unknown, but in some insects and plants the female meiotic spindle has an asymmetric distribution of microtubules or is monopolar [ 56 ], so a stronger centromere variant might better capture the favored pole. The new variant will therefore increase in the population and eventually become fixed. This meiotic drive process ('centromere drive') can account for the rapid evolution and complex structure of centromeric DNA. As a rare new variant spreads in the population, however, disparities in centromere strength may interfere with fertility in males, where the four meiotic products contribute equally to the next generation. Mutations in CenH3 that restore centromere parity in meiosis will therefore be selected in males, resulting in the adaptive evolution of CenH3 and suppression of the meiotic drive of centromeric DNA. Recurrent cycles of meiotic drive by centromere variants, or centromere drive, and suppression by CenH3 mutations would result in the observed rapid evolution of both centromeres and CenH3s. The lack of evidence for adaptive evolution in CenH3s from mammals and grasses does not seem to fit this scenario. But the extensive positive selection on the corresponding CENP-Cs provides a ready explanation for the absence of an adaptive signal for CenH3. The meiotic drive model predicts that over evolutionary time any mutation that restores centromere parity will be selected, suggesting that proteins besides CenH3 - and in particular other kinetochore proteins that contact centromeric DNA - may be positively selected to suppress centromere drive. Our demonstration of the adaptive evolution of CENP-C, especially in DNA-binding regions, fulfills this prediction of the centromere drive model. Apparently, in mammals and grasses CENP-C performs the function of a suppressor of meiotic drive. The large size and lack of sequence conservation of CENP-Cs make them much larger mutational targets for suppression than CenH3s. Moreover, PAML analysis suggests that a larger proportion of CENP-C than CenH3 residues are evolving adaptively. Mammalian CENP-A consists of a well-conserved histone-fold domain with only a short unconstrained tail region. Conversely, Drosophila species have the longest CenH3 tails known [ 12 ] but lack any identifiable CENP-C homologs. It is tempting to speculate that the interaction of the long CenH3 tail of Drosophila with centromeric satellites compensates for the absence of CENP-C and permitted its loss. This might explain why Drosophila CenH3s localize in a species-specific manner [ 44 ], whereas human CENP-A can be functionally replaced by its budding yeast CenH3 counterpart [ 57 ]. Centromere drive may have important consequences for karyotypic evolution. Centromeres of two acrocentric chromosomes frequently fuse (Robertsonian translocations), and metacentrics often misdivide to yield two acrocentrics. In humans, there is a bias in favor of Robertsonian translocations over their homologous acrocentric pair when transmitted by females, and male carriers have reduced fertility [ 58 ]. This general sterility of Robertsonian males is consistent with centromere drive underlying post-zygotic reproductive isolation in emerging species [ 1 ]. Centromere drive provides a mechanism for the tendency of karyotypes to be either mostly metacentric or mostly acrocentric [ 59 ] and for the karyotype-specific accumulation of selfish B chromosomes in mammals [ 60 ]. Our finding that CENP-Cs, like CenH3s, evolve adaptively addresses a perceived shortcoming of the centromere drive model for post-zygotic reproductive isolation: mutations that rescued hybrid sterility did not map to the Drosophila CenH3 gene [ 61 , 62 ]. The fact that CenH3 is not the only adaptively evolving centromere protein indicates that there are multiple candidate drive suppressors that might rescue hybrid sterility when in a mutant form. In contrast to CENP-Cs of plants and animals, yeast Mif2p appears to have evolved entirely under negative selection. This is consistent with Mif2p interacting with a stable centromere, rather than one that is rapidly evolving. In accordance with this observation, budding yeast centromeres are determined by the presence of a consensus DNA sequence that includes binding sites for the Cbf1 and CBF3 proteins [ 49 ]. The consensus DNA sequences and their binding proteins are recognizably similar in yeasts as distantly related as Candida glabrata and Kluyveromyces lactis , which have greater average divergence from budding yeast in protein sequences than mammals have from fish [ 63 ]. We attribute this extreme conservation of centromere sequence to optimization of the DNA-protein interactions at the centromere. Such optimization would be inevitable in fungi that produce equivalent gametes in a tetrad. No such optimization would occur when centromeres compete at female meiosis I for a favored orientation. Seed plants and animals evolved female meiosis independently, so the parallels that we see for evolution of CenH3 and CENP-C would reflect parallel evolutionary forces in these two ancient lineages. Materials and methods DNA clones and sequencing Genomic DNAs and cDNAs were obtained from several sources (Additional data file 2). The A. thaliana cDNA was amplified from a cDNA pool from whole plants of the ecotype Columbia, and the A. arenosa cDNA was amplified from a cDNA pool from leaves of Care-1 [ 64 ]. Both of these cDNAs were amplified using the same primers: 5'-GGAATTTTCCGGTGATTTAGATG-3', which terminates in the initiation codon, and 5'-TGATCACAAGAGGATGGTTGA-3', from the 3' untranslated region of the A. thaliana genomic Cenpc sequence. Genomic DNAs from wheat and barley were generously provided by Andreas Houben. Exons 9p-10p and the intervening intron 9p were amplified from both wheat and barley using the primers 5'-AGATGAACCAATCCATCCAC-3' and 5'-AAATTCGTTTTCCTCTCTTTGCT-3'. Likewise, 9q-10q and intron 9q were amplified with the primers 5'-AGATAAGCCAATCCATACATCA-3' and 5'-CCCCTCTTTTCATTCTCTTCAA-3'. The first and last of these four primers were also used to amplify both exon pairs as a unit to confirm their contiguity and to determine the genomic sequence around the junctions of intron 10p with exons 10p and 9q. Amplifications used High Fidelity Platinum Taq polymerase (Invitrogen, Carlsbad, USA). The amplified fragments were cloned using the pCR2.1-TOPO-TA cloning kit (Invitrogen) according to the manufacturer's instructions. Sequencing was carried out using ABI Big Dye sequencing on both strands of all reported sequences. Sequencing primers were standard vector primers or were designed using Primer 3 [ 65 ]. Sequences were assembled using Sequencher 4.1.2 software [ 66 ]. Accession numbers of sequences are given in Additional data file 2. Sequence analyses Sequence similarities of genes and their encoded proteins were identified using the NCBI BLAST server [ 35 , 67 ], as well as by use of Gramene [ 68 ] and the TIGR Gene Indices [ 69 ]. Translations and sequence manipulations utilized the Sequence Manipulation Suite [ 70 , 71 ]. Alignments of coding and amino-acid sequences were performed using the European Bioinformatics Institute Clustal W Server [ 72 , 73 ], with adjustments by hand to take account of splice-site alignment. Conservation in alignments was displayed using MacBoxShade 2.1 (MD Baron, Institute for Animal Health, Surrey, UK). Protein blocks were made, displayed, and compared using the Multiple Alignment Processor, sequence Logos, and LAMA [ 74 ] programs on the Blocks WWW Server [ 75 ]. To make blocks from grass exons 9-12, gaps in ClustalW alignments were first filled with Xs, which do not appear in subsequent sequence Logos representations. Gene models of exon-intron boundaries were made by alignment of cDNAs with identical or homologous genomic sequences, as well as by splice-site prediction using the NetGene2 server [ 76 , 77 ]. K-estimator [ 31 ] was used to estimate K a and K s in comparisons of Cenpa/CENH3 or Cenpc genes from pairs of closely related species. Prior to analysis, gaps were removed from the coding sequences as indicated by the amino-acid alignments. We estimated K a and K s for windows of 99 nucleotides, positioned every 33 nucleotides. For candidate regions of positive selection, we determined the confidence intervals of K a and K s under the null hypothesis that K a is equal to K s using the default parameters (1,000 replicates). For individual windows of 99 nucleotides, or for regions defined by contiguous groups of overlapping windows, limited trial and error suggested that statistically significant positive selection was not supported if K a /K s < 1.5. Therefore, to find evidence of positive selection, we determined the confidence intervals for regions defined by sets of overlapping or immediately adjacent 99 nucleotide windows with K a /K s ≥ 1.5. For regions with K s = 0, one or more flanking windows with K s > 0 were included in the region analyzed, regardless of the value of K a , so that a value for K a /K s could be defined. Similarly, we looked for statistically significant negative selection for regions defined by overlapping or adjacent 99 nucleotide windows with K a /K s ≤ 0.67. The codeml program of PAML version 3.13d [ 40 ] was also used to test for positive selection and to estimate K a /K s ratios as previously described [ 11 ]. Additional data files The following are provided as additional data files. Additional data file 1 , containing Table S1, reports accession numbers for selected Cenpc ESTs and genomic sequences from GenBank. Additional data file 2 , containing Table S2, reports accession numbers for Cenpc cDNAs and amplified genomic sequences. Additional data file 3 , containing Figure S1, displays the conservation of the exon containing the CENPC motif. Supplementary Material Additional data file 1 Table S1 reports accession numbers for selected Cenpc ESTs and genomic sequences from GenBank Click here for additional data file Additional data file 2 Table S2 reports accession numbers for Cenpc cDNAs and amplified genomic sequences Click here for additional data file Additional data file 3 Figure S1 displays the conservation of the exon containing the CENPC motif Click here for additional data file | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549713.xml |
549075 | HIV-1 Tat protein enhances Microtubule polymerization | Background HIV infection and progression to AIDS is characterized by the depletion of T cells, which could be due, in part, to apoptosis mediated by the extra-cellular HIV-encoded Tat protein as a consequence of Tat binding to tubulin. Microtubules are tubulin polymers that are essential for cell structure and division. Molecules that target microtubules induce apoptosis and are potent anti-cancer drugs. We studied the effect on tubulin polymerization of three Tat variants: Tat HxB2 and Tat Eli from patients who are rapid progressors (RP) and Tat Oyi from highly exposed but persistently seronegative (HEPS) patients. We compared the effect on tubulin polymerization of these Tat variants and peptides corresponding to different parts of the Tat sequence, with paclitaxel, an anti-cancer drug that targets microtubules. Results We show that Tat, and specifically, residues 38–72, directly enhance tubulin polymerization. We demonstrate that Tat could also directly trigger the mitochondrial pathway to induce T cell apoptosis, as shown in vitro by the release of cytochrome c from isolated mitochondria. Conclusions These results show that Tat directly acts on microtubule polymerization and provide insights into the mechanism of T cell apoptosis mediated by extra-cellular Tat. | Introduction AIDS is due to human immunodeficiency virus (HIV-1) infection of CD4 T cells and is characterized by the cell death of HIV-infected lymphocytes and uninfected bystander cells [ 1 - 4 ]. Recent discoveries have shown that this could be related to the extra-cellular effects of Tat, which is a toxic protein secreted early by HIV-infected cells [ 5 , 6 ]. Tat was first identified as a regulatory protein essential in the HIV viral cycle due to its ability to dramatically increase HIV gene expression [ 7 ]. The size of Tat varies and exists in forms between 86 and 101 residues. Yet 101 residues now appear to be the dominant size in the field [ 6 ]. Interest in this protein was raised with the discovery that Tat was secreted from HIV-infected cells and that it could have extra-cellular functions related to AIDS pathogenesis such as Kaposi's sarcoma [ 8 ]. The extra-cellular roles of Tat are suspected to be the major reason for the maintenance of HIV-infected cells (or reservoir cells) [ 5 ], and could explain the failure of current antiviral therapies to eradicate HIV [ 9 ]. Tat induces apoptosis in different cell lines such as macrophages and cytotoxic T-lymphocytes (CTL) [ 10 ], which are essential for the cellular response of the immune system to eliminate virus-infected cells [ 6 ]. Different mechanisms by which Tat induces apoptosis in T cells were proposed: (i) The up-regulation of Fas ligand [ 10 ]; (ii) The up or down regulation of cellular genes encoding for cytokines [ 11 ], for cell survival factors such as Bcl-2 [ 12 - 14 ], for superoxide-dismutase [ 15 ], and for p53 [ 16 ]; (iii) The inhibition of the expression of manganese-dependant superoxide dismutase [ 17 ]; (iv) The activation of cyclin dependant kinases [ 18 ]. Another mechanism was proposed that involves microtubules to induce Tat-mediated apoptosis [ 19 ]. We showed recently, using two short Tat variants of 86 residues, that Tat is able to form a complex with tubulin [ 20 ]. Microtubules are tubulin polymers necessary for the change and preservation of cellular morphology, intracellular organelle distribution, chromosome migration during mitosis, cell differentiation, as well as intracellular transport and signalization [ 21 ]. Microtubule damaging agents (MDAs) are classified in microtubule-stabilizing agents such as Taxanes and microtubule depolymerizing agents such as Vinca .alkaloids. The MDAs inhibit microtubule dynamics in living cells and lead to apoptosis after cell cycle disturbance [ 21 - 24 ]. They activate the intrinsic mitochondrial apoptotic pathway as shown by the mitochondrial membrane potential collapse and the opening of the permeability transition pore [ 25 , 26 ]. The subsequent release of pro-apoptotic factors, such as cytochrome c , leads to the caspase cascade activation and thus to apoptosis [ 27 ]. Moreover, MDAs can also directly affect mitochondria, as shown in vitro by the release of cytochrome c from isolated mitochondria [ 28 ]. The aim of this study was to evaluate the consequence of Tat binding to microtubules and the correlation with the mitochondria-mediated T cell apoptosis. We used three Tat variants with sequences from 99 to 101 that have the size of the main Tat variants found in the field [ 6 ]. The HIV-1 HxB2 and HIV-1 Eli are representative of rapid progressor (RP) patients in respectively Euro-American strains [ 29 ] and African strains [ 30 ]. HIV-1 Oyi strain was isolated from a highly exposed persistently sero-negative (HEPS) individual during an epidemiological study in Gabon [ 31 ]. In order to identify the regions of Tat essential for the interaction with microtubules, we used peptides corresponding to different part of the Tat sequence. We compared the effect of these Tat variants and Tat peptides with paclitaxel, a well-known anti-cancer drug agent that strongly increases tubulin polymerization and stabilizes microtubules [ 32 ]. Results Tat acts on tubulin in vitro The minimal concentration of tubulin (Cr) necessary to obtain tubulin polymerization without drug was 7 μM in our buffer conditions [ 33 ]. We measured the polymerization effect of various concentrations of Tat and paclitaxel with 15 μM tubulin. We did observe the enhancement of tubulin into microtubules in the presence of Tat and paclitaxel (Fig. 1A ). In comparison with tubulin alone (line 1), the rate of assembly as well as the final extent of assembly was enhanced by Tat. Furthermore, the lag time to start polymerization is shorter with Tat (lines 2 and 4) as compared with tubulin alone (line 1). With 4 μM (line 2) and 8 μM Tat (line 4), turbidity reached a plateau at 0.55 and 1.20 respectively, which corresponds to a 1.7 and 3.7 fold increase compared with the control plateau at 0.32 obtained with tubulin alone (line 1) (Fig. 1A ). With higher concentrations of Tat (16 μM and 20 μM) the turbidity plateaus were superior to 5. The lowest effective concentration of Tat that induced an increase in polymerization was 0.5 μM (data not shown). Figure 1 Turbidity time course of the in vitro microtubule assembly. ( A ) Tubulin in presence of various ligands. Samples with 15 μM tubulin are maintained at 4°C. After addition of 8 μM Tat (line 4), 4 μM Tat (line 2) from HIV-1 HxB2 strain or 15 μM paclitaxel (line 3), the assembly reaction was started by warming the samples at 37°C (time 0, arrow) and compared to tubulin alone (line1). The ΔOD350 nm is measured every 30 sec. After 30 min the temperature was lowered to 10°C (arrow). ( B ) Electron microscopy of microtubules formed in the presence of Tat. Aliquot from samples reaching the ΔDO350 nm plateau at 37°C were adsorbed on coated Formvar films on copper grids. Electron micrographs of microtubules formed without Tat (Control) or with indicated concentrations of Tat and paclitaxel are presented with 4000-fold magnification. Microtubules formed with Tat at 8 μM are also presented at 40000-fold magnification. ( C ) Production of microtubules in the presence of Tat. Samples of 15 μM tubulin alone (Control) and with 8 μM Tat (Tat 8), 16 μM Tat (Tat 16) or 15 μM paclitaxel (Paclitaxel) at the time they reach the plateau at 37°C were ultracentrifuged and supernatant (S) and pellets (P) were analyzed on SDS-PAGE. (Tat 8) × 5 indicates a fivefold increase in the quantity of the sample loaded. The mass of tubulin (Tub 55 kDa) and Tat (Tat 10 kDa) are indicated. When the temperature of the samples was decreased to 10°C, we observed a complete depolymerization for tubulin alone (line 1) and tubulin with 4 μM and 8 μM Tat (lines 2 and 4 respectively). Even at the highest concentrations of Tat (16 μM and 20 μM), at 10°C the turbidity decreased to the original values (data not shown). By contrast, in the same conditions, we did not observe any depolymerization in the presence of paclitaxel (line 3). However, full depolymerization in the presence of paclitaxel was obtained with a longer incubation at 4°C (data not shown). The reversibility of following an incubation at 10°C or 4°C strongly suggests that turbidity enhancement is not related to precipitation or aggregation but to microtubule formation. We also observed that when Tat concentration is increased to stoichiometric quantities with tubulin, the turbidity values increased dramatically to an absorbance greater than 2. These values are superior to the absorbance obtained in the same conditions using the stoichiometric quantity (15 μM) of paclitaxel, which induces effective tubulin polymerization. Turbidity is known to be a function of the total weight concentration of scattering particles only when the particles have small diameters compared with the wavelengths of the incident light [ 34 , 35 ]. Thus, it seemed Tat would be able to act quantitatively and qualitatively on tubulin polymerization. In order to address these possibilities, our first control was to verify the shape of microtubules formed in the presence of Tat. Examination with electron microscopy confirmed the formation of microtubules in the presence of Tat, that were similar in shape to the controls without Tat (Fig. 1B ). However, microtubules in the presence of Tat and paclitaxel were slightly packed and shorter than non-treated microtubules. These phenomena were in part responsible for the variation in turbidity. To determine whether a part of the enhancement of turbidity was due to an increase in tubulin polymerization, we did evaluate the amount of polymerized tubulin on gel after ultra-centrifugation. Results in figure 1C show that with Tat or paclitaxel the band intensity of pellets (P) are enhanced and the band intensity of supernatants (S) are decreased compared with the control tubulin alone. Increasing the concentration of Tat to 8 μM (Tat 8) and 16 μM (Tat 16) indeed enhanced the proportion of tubulin in the pellets (P) and decreased the proportion of tubulin in the supernatant (S) (Fig. 1C ). Thus, although the enhanced turbidity is due in part to microtubule aggregation, these results show that the increase expansion in the turbidity plateau is also due to an increasing amount of microtubules. Interestingly, analysis of the samples by gel electrophoresis, using a five-fold amount of the tubulin samples loaded on gels showed that Tat was only detectable in the pellet fraction (Fig. 1C ). To eliminate non-specific binding, we used a glycerol cushion during the ultra-centrifugation step [ 33 ]. This confirmed that Tat was associated with the microtubules. We evaluated the ability of different Tat variants to modify tubulin polymerization. In our conditions, when 15 μM of tubulin are incubated at 37°C with 8 μM Tat HxB2, Eli or Oyi, the turbidity plateau values at 350 nm are 1.01, 1.61 and 0.80 respectively (Table 1 ). These values are higher than the turbidity value of 0.30 obtained with 15 μM tubulin alone (Table 1 ). Thus Tat from different HIV subtypes are able to enhance tubulin polymerization. However, Tat Eli and Tat HxB2 are more effective in this enhancement than Tat Oyi (Table 1 , column O.D. ratio). Table 1 Enhancement of tubulin polymerization by different Tat and derived peptides Compounds Concentration ΔOD 350 nm ΔOD ratio none 0.31 1 Tat HxB2 8 μM 1.01 3.31 Tat OYI 8 μM 0.80 2.63 Tat ELI 8 μM 1.61 5.27 Paclitaxel 15 μM 0.55 1.79 Pep38–72 OYI 8 μM 0.78 2.56 Pep38–72 ELI 8 μM 1.59 5.20 Pep73–101 OYI 8 μM 0.31 1.01 Pep73–99 ELI 8 μM 0.30 1 15 μM tubulin is incubated at 37°C in buffer, the increase of ΔOD 350 nm is followed and the value of the plateau is indicated in the column ΔOD 350 nm . Lane 1 is the control with tubulin alone (none), lanes 2–4 are Tat variants, lane 5 is the paclitaxel control, lanes 6–9 are the Tat peptides. The ΔOD 350 nm ratio is the value that the ΔOD 350 nm plateau reaches in presence of the indicated product divided by the value of the ΔOD 350 nm plateau reached with tubulin alone. To identify the sequence(s) region(s) implicated in tubulin, peptides overlapping the sequence of Tat HxB2 and Tat Oyi were synthesized. The sequences of peptides 38–72 and 73–99 derived from both Eli and Oyi are indicated in figure 2 . Using 8 μM of peptide 38–72 from Eli (line Pep38–72 ELI) and Oyi (line Pep38–72 OYI) in the presence of 15 μM tubulin, we obtained a turbidity plateau value of 1.59 for Eli and 0.78 for Oyi which correspond to an increased ratio of 5.20 and 2.56 (Table 1 ). These effects were comparable with the effect of the entire parental Tat proteins. Turbidity with the peptide 73–99 derived from Eli (Pep73–99 ELI) and Oyi (Pep73–101 OYI) were at the same level as the control, indicating that they were not active in tubulin polymerization (last lines, Table 1 ). Figure 2 Sequences of HIV-1 Tat strains. These three Tat variants and the peptides were obtained by solid phase synthesis with a procedure previously described [43, 50]. Sequences correspond to the viral strains HIV-1 Eli [30], HIV-1 HxB2 [29] and HIV-1 Oyi [30]. Tat-mediated apoptosis in T-cells correlates with the effect on microtubules Drugs that are able to bind to tubulin and/or microtubules are known to affect the microtubule network organization and block the cell cycle in mitosis, leading to cell death. We first evaluated the toxicity of the different Tat variants on Jurkat lymphocyte cells, using 4'6-diamidino-2-phenylindole (DAPI) staining. This method allows the quantification of apoptotic cells by the observation of the characteristic nuclear fragmentation. After 20 hours treatment, the percentage of apoptotic cells in the presence of 10 μM Tat HxB2 (11 %) and 10 μM Tat Eli (12.5 %) were higher than those obtained with 10 μM Tat Oyi (3.5 %) (Fig. 3A ). These differences in apoptosis were significant ( p = 0.037) using one-sided ANOVA. The percentages of apoptotic cells in the presence of 1 μM Tat Oyi were similar to the control cells (0.1 %) (Fig. 3 ). The percentages of apoptotic cells in the presence of 1 μM Tat HxB2 (2.7 %), 1 μM Tat Eli (2 %) and 10 μM Tat Oyi (3.5 %) was weak and comparable to the basal level of the non-treated control cells (Fig. 3A ). Thus, at 10 μM Tat HxB2 and Tat Eli induced apoptosis inversely to 10 μM Tat Oyi. These results indicate that Tat-mediated apoptosis in T-cells could be correlate with the effect on microtubules since the differences in toxicity between the different Tat variants correspond to the same differences in tubulin polymerization. These differences were also observed using trypan blue exclusion (data not shown). Tat toxicity was then confirmed by flow cytometry analysis after propidium iodide (Pi) staining. Figure 3B shows that 1 μM and 10 μM Tat HxB2 induces 13% and 27% apoptosis respectively, revealed by the proportion of cells with hypodiploid DNA content (Fig. 3B , H values). A high proportion of apoptotic cells (31 %) was also obtained with 10 μM of Tat Eli. Furthermore, the percentage of hypodiploid cells obtained with 10 μM Tat Oyi is low (10.4 %) and is the same as the control without Tat (9.9 %). Figure 3 Effect of different Tat variants on cell cycle progression, apoptosis and microtubule network in lymphocytes. ( A ) Table showing data from fluorescence microscopy after 20 hours treatment with indicated concentration of Tat or paclitaxel. Percentage of apoptotic cells are determined after DAPI staining and the differences in apoptosis between treated and untreated cells used as control obtained in three independent experiments are presented. ( B ) Jurkat cells were treated with indicated concentrations of paclitaxel and various Tat or were untreated (control). After 20 hours, cells were stained with PI and analysed by flow cytometry. Percentage of cells in G2/M (G2 / M) or apoptotic cells (H) with hypodiploid DNA are indicated in the upper corner of each cell ( C ) Jurkat cells were treated with 10 μM Tat Eli or 1 μM paclitaxel and processed for immunofluorescence labeling with anti-alpha tubulin antibody as described in materials and methods. Control corresponds to untreated cells. Tat does not affect microtubule network organization nor cell cycle progression Paclitaxel stabilizes the microtubule network by inducing the formation of pseudoasters in mitotic cells and the formation of bundles, structures that correspond to strongly associated microtubules, in interphasic cells [ 21 ]. Considering that Tat increased microtubule polymerization, we investigated if Tat was able to induce bundle formation or another disturbance in the microtubule network organization in the treated cells. The effects of Tat and paclitaxel cytotoxic concentrations (10 μM and 1 μM respectively) were evaluated by immunofluorescence microscopy on Jurkat cells. After 6 hours treatment with paclitaxel, strong modifications of the microtubule network (that form bundles) were observed (Fig. 3C ). However, in the presence of 10 μM Tat, we did not observe bundles or any other modification of the microtubule network. By disturbing microtubule functions, MDAs generally lead to a mitotic block of treated cells. Although study have implicated the transactivation effect of Tat on cyclin and other gene implicated in cell cycle progression, we examined if Tat induced a mitotic block that could be due in part to the tubulin polymerization of Tat. Following treatment, the percentage of cells in various phases was obtained by flow cytometry analysis after propidium iodide incorporation. After 20 hours treatment, 1 μM paclitaxel blocks almost 50 % of the cells in the G2/M phase (Fig. 3B , G2/M values). This strong blockage in G2/M with paclitaxel attests that we were in the appropriate time conditions to evaluate the effects of Tat on cell cycle regulation in these Jurkat cells. A longer incubation time ( i.e. 48 h) in the presence of paclitaxel leads to 90 % of cell death (data not shown). After 20 hours treatment, the percentage of the G2/M cells in the presence of 1 μM Tat HxB2 or 10 μM Tat Oyi were 28 % and 32 % respectively and similar to the 33 % observed in the untreated cells (Fig. 3B ). In the presence of 10 μM Tat HxB2 or 10 μM Tat Eli the percentage of G2/M cells (22 % and 24 % respectively), were lower than the control cells (33 %). This decrease of the G2/M is not correlated with an increase in G1, and seems to be related to the strong cell death (30 %) rather than to a specific block in another cell cycle phase. In parallel, DAPI results confirmed that the percentages of metaphasic cells were similar between untreated cells and Tat-treated cells (1 %), in contrast to paclitaxel-treated cells that were blocked in metaphase (50 %). Thus, contrary to paclitaxel, the different Tat variants did not lead to a G2/M block before inducing apoptosis. Tat induces the release of cytochrome c from isolated mitochondria MDAs induce apoptosis through the mitochondrial apoptosis pathway. Our previous work on paclitaxel showed that this agent is able to induce cytochrome c release from purified mitochondria [ 28 ]. To investigate whether Tat could directly target mitochondria, we isolated these organelles and incubated them with growing concentrations of Tat Eli (0.2 μM, 2 μM and 10 μM). Using Western Blot analysis, we detected cytochrome c in the supernatant of mitochondria incubated with Tat Eli (Fig. 4A , Cyt c). Enhanced amounts of cytochome c were observed with 2 μM and 10 μM Tat Eli (lane T10 and T2) whereas the level of cytochrome c with 0.2 μM of Tat Eli (lane T0.2) was comparable to the untreated mitochondria (lane T-). In parallel, cytochrome c levels decreased slightly in mitochondria treated with Tat, as revealed by Western Blot of the pellets (Fig. 4B , Cyt c). Levels of VDAC, mitochondrial outer membrane porin, were systematically measured to ensure that equal amounts of mitochondria were present in the pellets (Fig. 4B , VDAC), and that no mitochondria remained in the supernatants (Fig. 4A , VDAC). Figure 4 Tat induces cytochrome c release from isolated mitochondria. After 2 hour treatment with 10 μM (P10) of peptide 73–99 or 0.2 μM (T0.2), 2 μM (T2) and 10 μM (T10) of Tat Eli or without peptide (T-), samples were centrifuged and supernatants and pellets were separated. ( A ). Western blots of the supernatant with antibodies against cytochrome c (Cyt C) and with antibodies against VDAC. ( B ) Western blots of Mitochondria Pellets. Data are representative of four independent experiments. Discussion Our in vitro study clearly shows that Tat directly interacts with microtubules and is able to enhance their formation. Turbidimetry tests showed that Tat strongly enhanced pure tubulin polymerization. The Tat concentration inducing tubulin polymerization is higher compared to the concentration of Tat in the plasma and it is possible that Tat concentration increases in the cell due to an active uptake similar to what is observed with paclitaxel. Furthermore, it is also possible that Tat concentration in the plasma is higher nearby HIV infected cells. We found that Tat acts qualitatively on microtubules making them shorter, such as paclitaxel, and also induces microtubule packing. These effects on microtubule formation are more pronounced than with paclitaxel as observed by turbidimetry and electron microscopy. The high effect of Tat on the self-association of tubulin is of interest for studies on the mechanism of microtubule formation and could be used in the design of new agents targeting microtubules. We also showed that the full-length Tat protein is not necessary to obtain tubulin polymerization enhancement as peptides derived from its central region harbored the same properties as the full-length protein. These peptides contain the glutamine-rich region of Tat and confirms that the glutamine-rich region of Tat is involved in Tat mediated apoptosis [ 20 ]. It also contains the basic region (sequence 49–60) that is essential for Tat to cross membranes [ 36 ] and the adjacent region (sequence 38–48) that has been previously shown to be involved in the Tat-tubulin interaction [ 19 ]. In this study, we demonstrate that the N-terminus and the C-terminus of Tat are not necessary for the interaction with tubulin. We showed that the more toxic Tat variants were also those that were more active in the polymerization of tubulin in vitro , suggesting that the toxicity of extra-cellular Tat is mediated by a mechanism involving microtubules. MDAs such as paclitaxel, which affect tubulin polymerization, generally disturb the microtubule network functions and inhibit cell proliferation. Contrary to paclitaxel, in the presence of high concentrations of Tat, we did not observe cell cycle arrest in G2/M. Moreover, by immunofluorescence microscopy, we did not detect any early modification of the microtubule network organization, even with high Tat concentrations. MDAs are able to induce apoptosis at lower doses than those required to induce bundles [ 21 ]. Interestingly, low concentrations of MDAs can suppress microtubule dynamics and disturb their functions, without modifying microtubule network morphology [ 21 , 27 , 37 ]. Thus, low cytoplasmic concentrations of Tat could modulate microtubule dynamics participating in the apoptotic signaling pathway induction in lymphocytes. The effect of Tat on microtubule dynamic needs to take in account that Tat could also modify the activity of proteins acting on microtubule dynamic such as LIS1 [ 38 ]. We hypothesize that Tat is able to induce the release of pro-apoptotic factors from mitochondria as we have previously shown this property for microtubule damaging agents [ 28 ]. We show here for the first time that Tat is able to directly affect isolated mitochondria and trigger cytochrome c release, a key event in the mitochondrial apoptotic pathway activation. This result is in agreement with those from Macho et al. that show that, in lymphocyte cultures under low serum conditions, Tat accumulates at the mitochondria. Moreover, this localization correlated with disruption of the mitochondrial membrane potential [ 39 ], a process that leads to the release of pro-apoptotic factors such as cytochrome c . Thus, we show in this study that secreted Tat can both act on tubulin polymerization and on the mitochondria in vitro . Interestingly, although our study involves only three Tat variants, our results show that, in comparison with Tat Oyi, the Tat HxB2 and Tat Eli derived from RP are more potent in tubulin polymerization and apoptosis induction in T-cell. This open the possibility that these properties of Tat could be associated with more severe disease phenotype. Apoptosis of bystander cells has been demonstrated to be very important in AIDS [ 40 ] and the rate of lymphocyte apoptosis has been correlated with progression rates [ 41 ]. It now appears that extra-cellular Tat secreted from HIV-infected cells is involved in the apoptosis of non-infected T cells. The elucidation of the mechanism responsible for Tat-mediated inhibition of the immune response therefore should have a tremendous impact for AIDS therapy. Methods Protein synthesis, Purification and Biochemical Characterization Tat variants and their derived peptides were synthesized with an ABI 433A peptide synthesizer (Perkin Elmer, Applied Biosystem Inc.) with FASTMoc chemistry according to the method of Barany and Merrifield [ 42 ] on 4-hydroxymethyl-phenoxy-methyl-copolystyrene-1% divinylbenzene preloaded resin (HMP; 0.50–0.65 mmol; Perkin Elmer, Applied Biosystem Inc., Foster City, CA), as previously described [ 43 , 44 ]. Purity and integrity of proteins were confirmed by amino acid composition (6300 Beckman analyzer), partial sequence analyses (473A Protein Sequencer, Applied Biosystem) and by MALDI-TOF mass spectrometry (Perspective Biosystems, Voyager DE-RP). Purified Lamb Brain Tubulin Lamb brain tubulin was purified by ammonium sulfate fractionation and ion-exchange chromatography, stored in liquid nitrogen and prepared for use as described [ 45 , 46 ]. During this purification particular caution was taken to remove microtubule-associated proteins (MAPs). Protein concentrations were determined spectrophotometrically with a Perkin-Elmer spectrophotometer Lambda 800. Tubulin extinction coefficient is ε 275 nm = 1.07 L·g -1 ·cm -1 in 0.5% SDS in neutral aqueous buffer or ε 275 nm = 1.09 L·g -1 ·cm -1 in 6 M guanidine hydrochloride. Tubulin polymerization In vitro studies have been derived from experiments that show that the variation of temperature permits the disassembly/reassembly cycles of tubulin in the presence of GTP and Mg 2+ [ 47 , 48 ]. Microtubule assembly was performed in 20 mM sodium phosphate buffer, 1 mM EGTA, 10 mM MgCl 2 , and 3.4 M glycerol, pH 6.5. The various concentrations of Tat or paclitaxel were mixed with tubulin at 4°C. The reaction was started by warming the samples to 37°C in a 0.2 × 1 cm cells, and the mass of polymer formed was monitored by turbidimetry at 350 nm using a Beckman DU7400 spectrophotometer thermostated. Depolymerization occurs when the temperature is lowered to 10°C. The data showed in fig 1a were representative of three independent experiments. ΔOD 350 nm values correspond to values without the buffer. The microtubule mass was determined by centrifugation assay. Samples were taken at the turbidity plateau at 37°C. They were ultra-centrifuged at 50,000 rpm for 15 min at 37°C using fixed angle rotor TLA 100.2 in a TL100 Beckman apparatus. In these conditions, microtubules precipitate in the pellet and non- polymerised tubulin remains in the supernatant. Pellet and supernatant were loaded separately on 10 % acrylamide SDS-PAGE, colored with Coomassie blue after migration (Fig. 1C ). Electron microscopy Small aliquots of tubulin associated with Tat were adsorbed on coated Formvar films on copper grids. They were negatively stained for 1 min in 2 % uranyl acetate and observed with a Jeol 1220 electron microscope. Cell Culture The lymhoblastoid Jurkat cell line was cultured in RPMI 1640 supplemented with 2 mM ultraglutamine (BioWhittaker, Verviers, Belgium), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10 % heat-inactivated fetal calf serum (BioWhittaker, Verviers, Belgium). Human neuroblastoma SK-N-SH cells were routinely maintained at 37°C and 5 % CO 2 , in standard culture RPMI-1640 medium (BioWhittaker, Verviers, Belgium) containing 10 % fetal bovine serum (BioWhittaker), 2 mM glutamine (BioWhittaker, Verviers, Belgium), 1 % penicillin and streptomycin (BioWhittaker, Verviers, Belgium). Exponentially growing cells (10 5 cells/ml) were seeded 3 days before paclitaxel or Tat treatment. Drugs and Antibodies Stock solution of paclitaxel (Sigma) was prepared in DMSO. The final concentration of DMSO used in cell culture was less than 0.05 %. For Western blotting, anti-VDAC antibody (anti-porin, 31 HL France Biochem, Meudon, France) was used at 1/500 and anti- cytochrome c antibody (7 h82C12; PharMingen, San Diego, CA) was used at 1/1000. Secondary antibody was goat antimouse monoclonal antibody conjugated with peroxidase (Jackson ImmunoResearch Laboratories, USA). The antibodies used for immunofluorescence microscopy were anti-α-tubulin (clone DM1A Sigma, Saint Louis, USA) and FITC mouse anti-goat IgG (Jackson ImmunoResearch Laboratories, USA). Cell death analysis Jurkat cells at a concentration of 5 × 10 5 cells/ml were incubated in the absence or presence of Tat or paclitaxel at different concentrations (see Fig. 3 ). After 20 hours incubation at 37°C, they were counted under optic microscope and cell death was estimated by trypan blue exclusion. Cells were then permeabilized with glacial alcohol, stained with propidium iodide and DNA content was measured by flow cytometry (Facs Calibur, Becton Dickinson, Mississauga, Canada). Cytogram analysis was performed with Cell Quest Pro ® software (Becton Dickinson, Mississauga, Canada). The proportion of hypodiploid cells was used as an estimate of apoptosis. Cell death was further analysed using fluorescence microscopy of cells stained with DAPI. 5 × 10 5 . Jurkat cells/ml were incubated at 37°C for 20 hours with various Tat or paclitaxel in Lab-Tek chamber slides. (Nalge Nunc International., USA). After centrifugation for 10 min at 3500 rpm, spin cells were fixed with 3.7 % formaldehyde, permeabilized with 0.1 % saponin and treated with PBS containing 10 μg/ml 4'6-diamidino-2-phenylindole (DAPI) (Sigma). The morphology of the cell nuclei was observed with a fluorescence microscope using an excitation wavelength of 350 nm. Nuclei were considered to have the normal phenotype when glowing bright and homogenously. Specific well drawn line up chromosome were seen in equatorial plates for cells in metaphase. Apoptotic nuclei were identified by the condensed chromatin gathering at the periphery of the nuclear membrane or a total fragmented morphology of nuclear bodies. Cells were counted and the percentage of normal., mitotic and apoptotic nuclei determined. Data indicated in the figure 3 are representative of three independent experiments. The apoptosis data obtained from the three independent experiments in the presence of paclitaxel and various Tat were compared using one-sided ANOVA following baseline subtraction using the values of untreated control cells. Indirect immunofluorescence staining of alpha-tubulin 10 6 Jurkat cells/ml were treated for 6 hours with 10 μM Tat HxB2 or 1 μM paclitaxel and were processed for tubulin indirect immunofluorescence staining as previously described [ 49 ]. After incubation with paclitaxel or Tat for 6 hr in lab-tek chamber slides (Nalge Nunc International, USA), the cells were centrifuged for 10 min at 3500 rpm. They were then fixed with 3.7 % formaldehyde and permeabilised with 0.1% saponin. Immunofluorescence microscopy of the microtubule network was performed using anti- α-tubulin antibody (Amersham, USA) and an FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, USA). Effect on mitochondria Mitochondria were isolated by cell fractionation from the neuroblastoma SK-N-SH cell line as previously described [ 28 ]. 9 × 10 7 cells were suspended in a sucrose buffer (250 mM sucrose, 1 mM dithiothreitol, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl 2 , phenylmethylsulfonyl fluoride, protease inhibitors, 20 mM Hepes, pH 7.4) at 4°C, homogenized with 50 strokes in a glass homogenizer (Kimble Kontes, Vineland, NJ), and centrifuged twice at 800 g for 10 min. The supernatants were then centrifuged at 15,000 g for 10 min at 4°C. Mitochondrial pellets were immediately washed three times in the sucrose buffer. Isolated mitochondria were aliquoted and incubated for 2 hr at 37°C with various Tat concentrations. After centrifugation (15,000 g for 10 min at 4°C), pellets and supernatants were carefully separated. Mitochondrial pellets were lysed in lysis buffer (62.5 mM Tris.HCl; pH 6.8, 0.5% SDS, 5% mercaptoethanol, 10% glycerol) and loaded in parallel with the supernatants on a reducing 15 % SDS-PAGE gel. The gel was processed for immunoblotting and revealed using antibodies against VDAC and cytochrome c. Anti-VDAC antibody (Calbiochem, USA) was used to ensure that equal amounts of mitochondrial proteins were loaded on the gels and that no mitochondria or lyzed mitochondria were present in the supernatant. Secondary antibodies were added and visualization was carried out using an enhanced chemiluminescence detection kit (ECL, Amersham, Aylesbury, United Kingdom). The results presented are representative of four independent experiments. List of abbreviations HIV, human immunodeficiency virus MDA, microtubule damaging agent VDAC, voltage-dependent anion selective channel. Competing interest The author(s) declare that they have no competing interests. Authors' contributions JdM carried out most of the experimental work, performed the experimental design and drafted the manuscript. MC performed part of the cell, mitochondrial and immunofluorescence study directed by DB. PB performed tubulin purification, part of tubulin experiments on polymerization, quantification of microtubules and electron microscopy directed by VP. GRC, SL, SO, DE and JW synthezised the peptides and characterized them biochemically and biophysically. CP performed FACS studies. EPL participated in the design of the study, the redaction of the manuscript and funded the studies. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549075.xml |
554971 | The pre-vaccination regional epidemiological landscape of measles in Italy: contact patterns, effort needed for eradication, and comparison with other regions of Europe | Background Strong regional heterogeneity and generally sub-optimal rates of measles vaccination in Italy have, to date, hampered attainment of WHO targets for measles elimination, and have generated the need for the new Italian National Measles Elimination Plan. Crucial to success of the plan is the identification of intervention priorities based upon a clear picture of the regional epidemiology of measles derived from the use of data to estimate basic parameters. Previous estimates of measles force of infection for Italy have appeared anomalously low. It has been argued elsewhere that this results from Italian selective under-reporting by age of cases and that the true measles force of infection in Italy is probably similar to that of other European countries. A deeper examination of the evidence for this conjecture is undertaken in the present paper. Methods Using monthly regional case notifications data from 1949 to the start of vaccination in 1976 and notifications by age from 1971–76, summary equilibrium parameters (force of infection (FOI), basic reproductive ratio ( R 0 ) and critical vaccination coverage ( p c )) are calculated for each region and for each of 5 plausible contact patterns. An analysis of the spectra of incidence profiles is also carried out. Finally a transmission dynamics model is employed to explore the correspondence between projections using different estimates of force of infection and data on seroprevalence in Italy. Results FOI estimates are lower than comparable European FOIs and there is substantial regional heterogeneity in basic reproductive ratios; certain patterns of contact matrices are demonstrated to be unfeasible. Most regions show evidence of 3-year epidemic cycles or longer, and compared with England & Wales there appears to be little synchronisation between regions. Modelling results suggest that the lower FOI estimated from corrected aggregate national data matches serological data more closely than that estimated from typical European data. Conclusion Results suggest forces of infection in Italy, though everywhere remaining below the typical European level, are historically higher in the South where currently vaccination coverage is lowest. There appears to be little evidence to support the suggestion that a higher true force of infection is masked by age bias in reporting. | Background The WHO target of eliminating indigenous measles in Europe by 2007 represents a challenge for public health systems. The requirements for success in this battle are summarised by Gay [ 1 ]. Italy, compared with other European countries, is still quite far from meeting these requirements. Here, in contrast with mandatory tetanus, polio, hepatitis B and diphtheria vaccinations, measles vaccination since its initiation in 1976 is only classed as 'Recommended' and has traditionally been characterised by very low coverage, with a national average in 12–24 months old children of only 56% in 1998 [ 2 ], and 76% in 2003 [ 3 ], despite intensified and supplementary efforts. A further worrying problem is persistent strong heterogeneity in coverage at the regional level (often also within each region). The substantial measles epidemics in Southern Italy in 2002–2003 [ 4 ] tragically underlined these points, also confirmed by routine and serological data [ 5 ]. In order to confront this state of affairs Italy is embarking on implementation of a Measles National Elimination Plan [ 6 ]. In designing an optimal elimination strategy, it is crucial that planners have at their disposal a clear picture of the regional "geography" of the intensity of effort required for measles elimination as a preliminary step for ranking intervention priorities. This is especially in view of the claim that there could be spatial heterogeneity in transmission rates due to the large socio-economical differences existing in the country [ 7 ]. However the evaluation of the required elimination effort in terms of critical coverages still largely and necessarily relies on estimates of basic reproduction numbers (also known as basic reproduction ratios) from pre-vaccination data [ 8 ]. Here we deal with this pre-vaccination epidemiology of measles in Italy, taking inspiration from two distinct standpoints. The first is a very practical one, i.e. the need to summarise the degree of effort needed for measles elimination in the Italian regions. This is carried out using the fundamental parameters of the basic SEIR (i.e. Susceptible-Exposed-Infected-Recovered) transmission dynamics mathematical model of vaccine preventable diseases[ 9 ]: forces of infection (FOI), defined as the per capita annual rate of infection among susceptible individuals), contact or 'Who Acquires Infection from Whom' (WAIFW) matrices (specifying the rates of transmission of infection between and within age groups) due to contacts arising from its own and from other age groups), and basic reproduction numbers ( R 0 , the mean number of secondary infections which would arise from the introduction of a primary infection in a wholly susceptible population). As primarily shown by Anderson & May [ 9 - 11 ], provided it is possible to estimate them from high quality pre-vaccination data, these parameters allow concise summary of the natural history of a given infection in a given country in the absence of vaccination, e.g. Edmunds et al . [ 8 ] for the epidemiology of measles, mumps and rubella (MMR) in Europe. Edmunds et al . [ 8 ] have shown that pre-vaccination patterns of measles (and mumps) in European countries were broadly similar, suggesting it may be possible to use parameter values estimated from other countries with good-quality pre-vaccination data to model measles (and mumps) in countries with no or poor infection data. The case of Italy was however more puzzling than that of the other countries. On the one hand they found that the FOI for measles (but also for mumps) computed from Italian national case notifications data was greatly different from other available European FOIs. On the other hand they computed the FOI implicit in the data of Santoro et al .[ 7 ] - the sole pre-vaccination measles sero-survey in Italy - finding figures that, at least for the youngest age groups (0–4), were in agreement with those summarising infection experience in European countries with good infection data, [ 8 ] - here referred to as "EURO" FOIs. They conjectured that Italian data suffered from strong selective under-reporting by age, and concluded: "It is tempting to dismiss FOI estimates from Italian case notifications data as the serological data are likely to be more robust". We believe, however, that this conclusion relies more on the generally assumed greater reliability of serological data compared to case reports, rather than on a full demonstration. Indeed, the FOI they estimated from the data of Santoro et al . [ 7 ] for school age children (age 5–10) is just 50% of the corresponding EURO FOI, i.e. even smaller than that estimated from case reports. We believe therefore that it would be prudent and worthwhile to try to obtain further insight into this problem. The Edmunds et al . [ 8 ] paper provides therefore the motivation for our second, more theoretical, question of whether or not the true FOI acting in Italy during the pre-vaccination era is homologous to the EURO FOI, as they suggest? The implications are relevant especially for the purposes of modelling which may be used to inform policy: is it advisable simply to rely on this EURO FOI, or does prudence dictate that one should consider also other possibilities? In what follows we use the term "EURO conjecture" to denote the suggestion of Edmunds et al .[ 8 ] that i) the Italian FOI would indeed be essentially homologous to the EURO FOI, and ii) Italian case notifications data simply camouflage this fact thanks to broad selective under-reporting. To shed more light on these issues, we have analysed more deeply Italian case notifications data by looking at patterns at several spatial levels. First we have systematically looked at the structure of the FOIs (and contact patterns, basic reproduction numbers, etc) for all the Italian Regions and Provinces. Since the national datum used by Edmunds et al . [ 8 ] to compute the Italian FOI, was obtained by pooling regional data in presence of strong spatial heterogeneity in under-reporting (documented in Williams et al . [ 12 ]), a deeper investigation of spatial patterns of infection could reveal the existence of some between regions heterogeneity in age-related transmission rates that could be an indicator of a higher force of infection (or indeed perhaps suggest the presence of selective under-reporting by age). It is indeed quite conceivable that true heterogeneity might exist within Italy as a result of contrasts between North and South in terms, for example, of family size (including that of the extended family), patterns of shared childcare and schooling, impact of climate on time spent indoors and out, etc.; however in the absence of systematic community based investigation such ideas must remain in the realms of speculation. Second, we carried out a systematic time series analysis of measles periodicities in the Italian regions during the pre-vaccination era (Fig 1 & 2 ). Our feeling here is that only under exceptional circumstances can selective under reporting by age mask true time patterns of incidence. Figure 1 Measles cases notifications in Italy. The regionally heterogeneous monthly pattern of Italian measles cases reported for the period 1949-1996 (NB Two time series are shown for Friuli as the city of Trieste was not incorporated into the region until its post-war status within Italy was resolved in 1954) Third, we add some results from our modelling work on measles in Italy. The results suggest that i) forces of infection estimated from case reports of measles in all the Italian regions are systematically and significantly lower compared with the EURO FOI with little, or only moderate, between regions heterogeneity; ii) regional periodicities mostly suggest a longer (usually 3 years or more) inter-epidemic period compared to England & Wales, a fact which is consistent with lower transmission (and which, together with the lower FOIs, suggests lower vaccination coverage might suffice to achieve control and elimination); iii) predictions from a mathematical model using the EURO force of infection, even compared with those based on a FOI estimated from Italian case reports, poorly match the 1996/1997 Italian serological data [ 13 ]. Methods The geographic analysis of the epidemiology of vaccine preventable diseases is made difficult by complex correlations between local dynamics. A first, though clearly partial, step is characterization of local infection patterns by treating spatial sub-areas as autonomous epidemiological units. Two measures are used here to characterise the regional (i.e. local) "landscape" of measles in Italy in the pre-vaccination era: summary parameters from the SEIR model for vaccine preventable disease, and summary periodicities from time series analyses of measles incidence. Age patterns of infection, reproduction numbers and critical vaccination coverage From the pre-vaccination age distribution of cases we computed, for each region, summary equilibrium parameters from the SEIR model: i) forces of infection, FOI; ii) mixing or contact ("who acquires infection from whom", WAIFW) matrices; iii) basic reproduction numbers, R 0 ; and iv) related critical vaccination coverages, p c , the proportion of a population needed to be successfully immunised at age zero with a 100% effective vaccine in order to eliminate the infection, where p c = 1-1/ R 0 (i.e. whereas in a wholly susceptible population one primary case will on average successfully transmit to R 0 contacts, in the case when the proportion already immune is greater than p c transmission will succeed for less than R 0 (1- p c ) = R 0 (1- [1-1/ R 0 ]) = 1.0 contacts) [ 9 ]. For comparison purposes we used the same age groups as in Edmunds et al . [ 8 ], i.e. 0–1 years, 2–4 yr., 5–10 yr., 11–17 yr., 18+ yr. The basic reproduction numbers [ 14 ] were computed for several plausible mixing matrices. Mixing (WAIFW) matrices Mixing matrices (Table 1 ) are used in standard infectious disease modelling [ 9 ] to summarise age patterns of contacts between susceptible and infected individuals; the generic element β ij summarises the risk of acquiring infection for a susceptible individual in age group i due to contacts with infective individuals aged j . When estimating from age-structured data (e.g. serology or case reports), a mixing matrix with m age groups can have at most m distinct elements [ 9 ]. In the simplest type of mixing, i.e. homogeneous mixing, the β ij entries are independent of age ( β ij = β for all i,j ). Other types of mixing matrix are considered in the paper and are listed below. However not all forms of mixing are compatible with a given force of infection. As noted by Anderson & May [ 9 ], a mixing matrix can be non feasible for the given FOI (i.e. it may yield negative values for some of the β coefficients) and in such circumstances "the chosen matrix is inappropriate to the observed age dependence in the FOI". Hethcote [ 15 ] further pointed out that not all feasible mixing matrices are "acceptable", in that they could lead to numbers of contacts between individuals outside plausibility bounds; inspection of the ensuing mixing matrices is thus necessary to avoid trivial results. Hethcote also proposed a "plausibility criterion", based on a postulated "preference for assortativeness", which is simple to use for preferred matrices (type PREF below). Table 1 Some of the types of mixing matrices used in the paper. Three of the mixing matrices discussed in Methods: a) fully assortative mixing (RDIAG), b) "Realistic" assortative mixing (DIAG) and c) the default mixing matrix (DEF) of Edmunds et al (2000). Succeeding rows and columns represent the age groups 0–1 year, 2–4 years, 5–10 years, 11–17 years and 18+ (e.g. for DIAG β 3 is the element corresponding to contacts between those in the 5–10 year age group and their peers in the same age group) a) DIAG b) RDIAG c) DEF β 1 0 0 0 0 β 1 β 5 β 5 β 5 β 5 β 1 β 1 β 1 β 1 β 5 0 β 2 0 0 0 β 5 β 2 β 5 β 5 β 5 β 1 β 2 β 4 β 4 β 5 0 0 β 3 0 0 β 5 β 5 β 3 β 5 β 5 β 1 β 4 β 3 β 5 β 5 0 0 0 β 4 0 β 5 β 5 β 5 β 4 β 5 β 1 β 4 β 5 β 3 β 5 0 0 0 0 β 5 β 5 β 5 β 5 β 5 β 5 β 5 β 5 β 5 β 5 β 5 Matrix types considered 1) Fully assortative mixing (matrix DIAG, Table 1(a) ) Individuals of a given age are assumed to mix only with individuals of the same age, yielding a diagonal matrix. Given a specific FOI it is the form of mixing allowing R 0 to achieve its upper bound [ 16 ]. 2) Realistic assortative mixing (RDIAG, Table 1(b) ) This matrix (termed "Diagonal" in Edmunds et al . [ 8 ], see also [ 17 ]) has the same diagonal elements as the fully assortative one, but some mixing across age groups is also possible (i.e. non diagonal elements are greater than zero). Here mixing across age groups is assumed to be at the same rate ( β 5 ) as that between adults [ 8 ]. 3) Default mixing (matrix DEF in Table 1(c) ) This matrix emphasises transmission between school age groups [ 8 ]. 4) Proportionate mixing (PM) Under PM [ 15 ] contacts occur at random, thus implying a larger probability of meeting more socially active individuals. The entries of the PM matrix have "multiplicative" form β ij = b i b j j = 1,... m . 5). One-parameter preferred mixing (PREF) The PREF matrix is a single parameter ( h ) weighted average of proportionate (PM) and fully assortative (DIAG) mixing: PREF = (1 - h ) * DIAG + h * PM , 0< h <1 [ 15 ], representing a contact pattern which can be split into a selective (i.e. non random) component (here mixing with individuals of the same age) and a random one. The PREF matrix has ( m+1 ) distinct entries: the extra parameter h is usually estimated "ad hoc". The matrices RDIAG & DEF are defined " ad hoc ", though they have a behavioural basis, whereas DIAG & PM are limit cases (though in distinct senses), and hence PREF is a weighted average between two limit cases. There is thus no clear relationship between, for instance, RDIAG & DEF on the one hand and PREF on the other. For this reason, we explored all forms that have been reported in the paper. Periodicities of time series of measles incidence In contrast to the spatially well synchronised biennial England & Wales oscillation, visual inspection of Italian regional data did not suggest clear common patterns of oscillation or hence of the inter-epidemic period. Thus investigation of periodicities in regional measles incidence became necessary to provide satisfactory characterisation of the inter-epidemic period. Though incidence data might be seriously affected by under-reporting, recent work [ 12 ] suggests that, as long as we are concerned with the pre-vaccination period, overall under-reporting rates in the Italian regions seem to have remained fairly constant over time. Thus the available time series should nonetheless represent a sufficiently reliable picture of the regional dynamics of measles. The cyclical behaviour in the monthly incidence time series of measles for each Italian region in the pre-vaccination period 1949–1976 was analysed in the frequency domain (first used for childhood diseases by Anderson et al . [ 18 ]) with special attention being given to the long term cycle. A drawback of periodogram analysis is the assumption that cyclical components of frequencies depend on the length of the observed series, i.e. that they are integer multiples of 2π/T ( T = series length); this is the basis of "harmonic" analysis. Such analysis may not identify exactly cyclical components where true frequency falls between two "harmonic" frequencies, e.g. it may suggest periodicity of either 3 or 4 observations when true periodicity lies between these values and hence is not identifiable exactly with this procedure. This problem may be overcome with "non-harmonic frequency domain" analysis where dependence of periodicities of cyclical components on series length is relaxed. Here the following procedure, as proposed in [ 19 ], is used for identifying true cyclical components: after log transformation to stabilise variances, and de-trending, using a deterministic function of time, the true frequency of a cyclical component, denoted by λ, was estimated, as in [ 19 ], by minimising, with respect to λ , the quantity where x t denotes the detrended series (a starting value for λ was obtained by examining a non-parametric estimate of the spectral density function; significant non-harmonic functions can be estimated in this way and deleted, if required, in a stepwise manner beginning with the frequency corresponding to the largest spectral density ordinate). The results from the time series analysis were also compared with the prediction from the homogeneous mixing SEIR model that where the sum K of the expected duration of the latent and infectious states is short compared to the life of the host (as is the case with measles), disease incidence will have a long-term oscillation around its endemic equilibrium with the period given, to an excellent approximation, by [ 9 ], where A is the average age at infection and K can be taken to be 14 days in the case of measles. Analyses described above were carried out for all Italian regions (Valle d'Aosta, a very small northern Region with few reported cases per year was aggregated with neighbouring Piemonte). Data In addition to published official Italian data on births, and birth and death rates, monthly measles case reports were provided by the Istituto Nazionale di Statistica (ISTAT) for the period from the first available year, 1949, to 1976, together with regional age structured measles case reports from the first available year, 1971. Pre-vaccination FOIs at the regional level were estimated from data for the time window 1971–76, which encompassed about two full three-years long epidemic cycles (inclusion also of the first few years in the post-vaccination window, when vaccine uptake was known to be extremely small, resulted in no significant change). Results: the pre-vaccination landscape of measles in Italy Regional patterns of incidence over time The spectral densities of the pre-vaccination (1949–76) Italian regional time series of measles incidence indicate, besides a well-pronounced annual cycle, the presence of a less pronounced longer term cycle of varying length, which is in contrast with the sharp biennial oscillation in England & Wales. Given the importance of the long term oscillation which is taken as representing the true "inter-epidemic period", the non-harmonic estimator was calculated (after de-trending each series using a function of time, and de-seasonalising, using monthly dummies,). Table 2 reports for each region: i) the average age of cases in the pre-vaccination period ( A ); ii) the non-harmonic estimate T O of the period of the long-term oscillation; iii) the length of the inter-epidemic period from the homogeneous SEIR model via formula for T given in the Methods section. Table 2 Average age at infection ( A 15 ) and inter-epidemic period (T) in the Italian regions. Results arising from the time series analysis of regional measles notifications data for the period 1949–76 (N = North, C = Centre, S = South.) A 15 (years) T from non harmonic estimate (T O ) (years) T from SEIR model (T) (years) Piemonte & Valle Aosta (N) 6.34 2.39 3.08 Lombardia (N) 5.70 2.37 2.94 Trentino (N) 5.75 2.85 2.94 Veneto (N) 5.87 3.21 2.98 Friuli (N) 5.98 2.77 2.98 Liguria (N) 6.92 5.34 3.22 Emilia (N) 6.24 3.33 3.06 Toscana (C) 7.30 2.85 3.29 Umbria (C) 6.81 3.40 3.21 Marche (C) 6.63 3.27 3.16 Lazio (C) 6.34 3.23 3.08 Abruzzo (S) 6.46 3.40 3.13 Molise (S) 6.19 5.88 3.04 Campania (S) 5.64 3.03 2.90 Puglia (S) 5.20 3.79 2.79 Basilicata (S) 5.43 3.74 2.82 Calabria (S) 5.63 3.45 2.90 Sicilia (S) 5.47 3.14 2.85 Sardegna (S) 5.70 3.79 2.92 North 6.09 2.35 3.02 Centre 6.78 3.27 3.22 South 5.65 3.68 2.90 Italy 6.18 2.35 3.06 Values of T O in table 2 show that, apart from a few Northern regions with a long term cycle with a period below 3 years (the shortest period, 2.4–2.5 years, being observed in Piemonte and Lombardia), most Italian regions have a three-year, or greater, long term oscillation (though a note of caution must be sounded with regard to the 5 year period observed in Molise, a very small isolated region). In comparison, values of T predicted by the SEIR model range from a minimum of 2.8 (Puglia) to 3.3 years (Tuscany). The agreement between T and T O in some cases is not very good: North-Eastern regions show a higher average age at infection, A , compared to Southern regions and yet the shorter inter-epidemic periods in the former compared with the latter, as suggested by the time series analysis, would imply the reverse. Compared to England & Wales there is also a surprising lack of synchronisation between regional cycles in the pre-vaccination period. A cross-spectral analysis suggests limited correlation between pre-vaccination long term cycles; coherencies greater than 0.7 were observed, as expected, only for a few neighbouring regions such as Emilia-Romagna and Marche, Marche and Umbria, Friuli and Trentino, Lombardia and Piemonte, Liguria and Piemonte The structure of the force of infection at the regional level Pre-vaccination age-distributed forces of infections in the Italian regions (we report a sample of results) can be well summarised by 3 clusters, North, Centre, South. These are shown in Fig. 3 together with the EURO FOI [ 8 ] which displays a similar qualitative pattern with age (single-humped, peaking in the "elementary school" age group 5–10, etc.), although the "Italian" FOIs exhibit surprisingly lower levels amongst pre-secondary school (< 11 years) age groups (with the exception, perhaps, of the very youngest age group in Southern Italy). Of the three Italian clusters the FOI for the two youngest age groups is highest in the Southern regions, and lowest in the Central regions. Relatively lower FOIs persist in Central regions in the elementary school age group (5–10 y.) compared with those in the North and South which resemble each other. Finally, in the two highest age groups, FOIs are similar, though Central and Northern regions now have marginally higher FOI values than the South. Figure 2 Measles cases notifications in Italy. Annual age distributions of measles case reports from 1971 (the first year in which notifications were recorded by year of age rather than age band) through to 1986 (10 years after the start of measles vaccination in 1976, albeit at very low coverage). The average age at infection A (computed over the restricted support 0–18 yr. age group, as more robust than the overall average age), that coarsely summarises the force of infection, shows similar spatial patterns. For instance A is systematically smaller in the South (little above 5 years), around 7 years in the Centre, and takes intermediate values in the North (Table 2 , second column). From the regional FOIs we also computed a national FOI corrected for under-reporting ('Italy-UR' in Table 3 ) using the under-reporting factors estimated in Williams et al .[ 12 ] which suggested great heterogeneity in reporting rates, the higher rates being observed in the Northern Italian regions (around 10–12% in the pre-vaccination era) and the smaller in the South (as low as 2% in Campania!). Conceivably the concurrence of spatial heterogeneity in overall rates of under-reporting with spatial heterogeneity in age-related transmission rates might be a factor responsible for a selective age-bias in the national age distribution even in absence of selective under-reporting by age, because it weights incorrectly the regional cases that are pooled into the national datum. Fig. 3 suggests however that the quantitative impact of the correction for under-reporting is likely to be rather small. Table 3 Measures indicating the size of the task of eliminating measles in the Italian regions. Estimates for basic reproduction numbers ( R 0 ) and critical age zero routine vaccination coverages ( p c ) for measles elimination in the Italian regions. Results in column pairs correspond to different assumptions on contact patterns. (NA = contact matrix non admissible; * = contact matrix admissible only after FOI redefined on adult age group; N = North, C = Centre, S = South.) Type of mixing pattern Homogeneous Default Realistic assortative Proportionate Preferred mixing ε = 0.9) R 0 P C age 0 R 0 P c age 0 R 0 P c age 0 R 0 P c age 0 R 0 P c age 0 Piemonte & Valle D'Aosta (N) 11.83 0,92 NA NA 11.8* 0.92 4.09 0.76 4.30 0.77 Lombardia (N) 13.16 0,92 6.2 0.84 16.1* 0.94 4.80 0.79 5.18 0.81 Trentino (N) 13.05 0,92 5.4 0.81 16.0* 0.94 4.50 0.78 4.82 0.79 Veneto (N) 12.77 0.92 5.5 0.82 17.6* 0.94 4.77 0.79 5.13 0.81 Friuli V.G. (N) 12.53 0.92 4.9 0.80 13.1* 0.92 4.05 0.75 4.32 0.77 Liguria (N) 10.83 0.91 NA NA 10.0* 0.90 3.73 0.73 3.95 0.75 Emilia (N) 12.01 0.92 NA NA 17.5* 0.94 4.41 0.77 4.69 0.79 Toscana (C) 10.27 0.90 NA NA 9.8* 0.90 3.60 0.72 3.79 0.74 Umbria (C) 11.01 0.91 NA NA 8.5* 0.88 3.62 0.72 3.81 0.74 Marche (C) 11.32 0.91 NA NA 10.6* 0.91 3.83 0.74 4.06 0.75 Lazio (C) 11.83 0.92 NA NA 11.8* 0.92 4.06 0.75 4.27 0.77 Abruzzo (S) 11.60 0.91 NA NA 14.6* 0.93 4.64 0.78 5.07 0.80 Molise (S) 12.11 0.92 NA NA 13.2* 0.92 5.68 0.82 6.62 0.85 Campania (S) 13.30 0.92 7.5 0.87 16.3 0.94 5.91 0.83 6.89 0.85 Puglia (S) 14.43 0.93 7.80 0.87 20.1 0.95 6.01 0.83 7.14 0.86 Basilicata (S) 13.82 0.93 6.7 0.85 19.8 0.95 5.19 0.81 5.86 0.83 Calabria (S) 13.32 0.92 6.1 0.84 18.5 0.95 5.07 0.80 5.62 0.82 Sicilia (S) 13.71 0.93 7.5 0.87 16.6 0.94 5.91 0.83 6.98 0.86 Sardegna (S) 13.17 0.92 6.3 0.84 16.6 0.94 5.29 0.81 5.93 0.83 North 12.32 0.92 5.2 0.81 14.9* 0.93 4.38 0.77 4.66 0.79 Centre 11.05 0.91 NA NA 10.4* 0.90 3.76 0.73 3.96 0.75 South 13.27 0.92 6.6 0.85 16.4 0.94 5.45 0.82 6.19 0.84 Italia 12.14 0.92 5.2 0.81 13.2* 0.92 4.33 0.77 4.64 0.78 Italy-UR 12.93 0.92 6.0 0.83 14.3 0.93 5.04 0.80 5.57 0.82 EURO 17.05 0.94 9.6 0.90 29.3 0.97 7.14 0.86 8.95 0.89 A further point worth of consideration in Fig. 3 is that, in contrast to the EURO FOI, in the North and Centre the FOI in the highest age group is higher than the FOI in the youngest (a fact shared by most regions in the two clusters, but not occurring in the Southern regions and which occurs regardless of how we define the last age group). This suggests a greater relative importance of contacts between adults (provided one can exclude the effects of poor reporting). Mixing matrices, reproduction ratios and required effort for measles elimination From the values of the FOI we computed mixing-WAIFW matrices. As previously noted, a problem with the computed Italian FOIs is that risk of infection among adults individuals (i.e. age group 18+) is, with the exception of Southern regions, higher than in the "young" (0–1 yr.). A verifiable consequence of this fact, is that "realistic assortative" mixing matrices (RDIAG) can be non-feasible (they can yield negative coefficients), and this indeed happens in all Central and several Northern regions. In other words, such mixing is not compatible, Southern Regions apart, with observed forces of infection. This may be a problem because "realistic assortative" is the mixing pattern that provides under a EURO-type FOI, the upper bound of "plausible" values of R 0 , a measure of critical importance from the perspective of disease control. For purposes of comparability with Edmunds et al . [ 8 ] we therefore arbitrarily redefined the value of the force of infection in the oldest age group in order to recover, for all Italian regions (and not only the Southern ones) the "EURO" shape (i.e. a FOI having its smallest value in the adult age group). In this manner we obtained feasible and "plausible" "realistic assortative" mixing matrices for all Italian Regions. The results are summarised below (Table 3 gives a synoptic view; we have omitted for sake of brevity the outputs of mixing computations and instead report the more easily interpretable values of reproduction ratios and critical vaccination coverages); the results from "fully assortative mixing" are omitted as leading to trivially high values of R 0 : 1. Homogeneous mixing Values of R 0 (computed as R 0 ≅ L / A where L is the expectation of life, taken as 75 years by assuming type 1 mortality and ignoring regional variability, and A the average age at infection in the pre-vaccination era, Table 2 ) range between a minimum of about 10 (Toscana) up to a maximum around 14.5 in Puglia (17.5 under EURO). 2. Realistic assortative mixing (RDIAG) This mixing yields an R 0 around 29 under EURO [ 8 ]. Thanks to our correction for the adult age group all Italian Regions yield fully admissible transmission rates; the corresponding R 0 values are in the range 13–20 in the South (critical coverages ranging 95–97%) and 8.5–12 in the Centre (critical coverages 88–92%), with intermediate values in the North. 3. Default mixing (DEF) It happens that this mixing pattern is never admissible for all Central Regions and for some Northern ones, as it can yield negative transmission rates when, as is the case in Central Italy, the FOI is rather low in the youngest age groups. Default mixing is, on the contrary, admissible in all Southern regions where R 0 and critical coverages happen to be systematically higher. Critical coverages range from 80% in the North up to 88% in the South (the reference EURO value being 90%). 4. Proportionate mixing (PM) PM mixing matrices (always admissible by definition) are the type of mixing [ 15 , 17 ] yielding the lower bound of "plausible" values of R 0 . Predicted critical coverages ranged from 72% in the Centre, with intermediate values in the North, up to 83% in the South (EURO = 86%, Table 2 ). Nevertheless inspection of the matrices does reveal for all regions (also for EURO) the presence of implausible relationships between groups, e.g. a significant level of disassortativeness. 5. Preferred mixing (PREF) We computed preferred matrices by tuning the "preference for assortativeness" parameter h starting from h = 1 (proportionate mixing) and then [ 15 ] progressively decreasing h until "minimally plausible" contact rates were achieved. Essentially, for all Italian regions (also for EURO), use of a moderate degree ( h = 0.9 ) of assortativeness allowed the "minimal plausibility" threshold to be surpassed. This led, compared to the case of proportionate mixing, to increases of 1% to 3% in the corresponding critical coverages (Table 3 ). These figures should be considered as providing more reliable lower bounds on R 0 than does Proportionate mixing. An issue is the degree of assortativeness, h , which would lead to the preferred mixing pattern closest to reality (here we only considered the degree guaranteeing a "minimally plausible" contact pattern, according to Hethcote [ 15 ]). Although one-parameter preferred mixing provides a flexible family of mixing patterns, it is still too inflexible to provide realism, as it assumes the same degree of preference for assortativeness in all age groups. For instance considering schooling as the major source of assortativeness in younger age groups, in pre-vaccination Italy a much smaller proportion, compared to the present, of boys in the youngest age group were attending crèche schools so that a major potential source of their assortativeness was probably absent. This would therefore suggest that at least two "preference for assortativeness" parameters ( h 1 , h 2 , say) may be needed to describe Italian pre-vaccination infection patterns. This also leads to the nub of the real gain provided by preferred matrices (just because they are "many") compared to "ad hoc" behavioural matrices. One could ask whether Hethcote's [ 15 ] preference for an assortativeness criterion is meaningful for mixing matrices which do not conform to the one-parameter "preferred mixing" scheme. Using this criterion of "preference for assortativeness", in most cases, "default" mixing would be discarded, and indeed, under default mixing (quite apart from the over small degree of assortativeness postulated for the youngest age group), most Italian regions do not meet this criterion for age groups 2–4 and 5–10. Nevertheless, from other perspectives, "default" is one of the most "reasonable" among behavioural mixing matrices. More generally, any answer to such questions is complex, as the problem of how to assess whether one arbitrary given mixing matrix is "better", or simply more plausible, than another remains unresolved. This problem is not simply an academic one: the predicted critical coverage for measles at age zero under the EURO FOI ranges between 86% under proportionate mixing (PM) and 97% under realistic assortative (RDIAG) [ 8 ]. Using the Hethcote [ 15 ] criterion one can marginally increase the lower bound up to 87.5% by replacing proportionate mixing (unacceptable under Hethcote criterion) with the corresponding "minimally acceptable" preferred matrix (PREF), but the extent of the uncertainty that remains is hardly less significant. A modelling result Our analysis of the pre-vaccination force of infection of measles in Italy formed a step toward developing a mathematical model for the transmission dynamics of measles in Italy. The model has the following features: i) it provides bounds for the uncertainty surrounding the estimate of the FOI (Table 4 ), by taking the "EURO" FOI as upper bound, and as a lower bound the FOI (denoted as Italy-UR in Table 2 & Figure 3 ) estimated from Italian case reports by correcting regional figures using the estimates of under-reporting in Williams et al . [ 12 ]; Table 4 Force of infection estimates. Estimates of EURO force of infection (FOI) and that from Italian case notifications; computations were based on standard techniques described by Anderson & May [9]. Force of infection/FOI (% / year) Age group (years) 0–1 2–4 5–10 11–17 18+ Italy-UR 7 15 31 19 6 EURO 12 28 40 20 10 Figure 3 Pre-vaccination forces of infections in the Italian regions. Age-related pre-vaccination forces of infection (FOI) estimated from case notifications for North, Centre and South divisions of Italy compared with the "EURO" FOI estimated by Edmunds et al [8] ii) it incorporates "realistic" demography in order to mirror the broad process of population ageing observed in Italy in the past 20 years (and which would be observed in the future if the present state should continue) as a consequence of the onset of sustained low fertility; iii) it considers two distinct options as regards interaction between the demography and the epidemiology: the first (D1) assumes no effects on transmission arise as a consequence of population ageing; the second (D2) assumes low fertility and population ageing could lead to a significant decline in transmission (e.g. a decline in the FOI), via proxy mechanisms such as a substantial decline in intra-family transmission which might occur as a consequence of contraction in average family size. By crossing the two FOI assumptions, EURO and UR (i.e. Italy-UR), with the two assumptions D1, D2 we obtain four assumptions: EURO/D1, EURO/D2, UR/D1, UR/D2. The model simulations were carried out by adopting for the post-vaccination era what we considered the "best" approximation for the profile of routine vaccination coverages (Fig. 4 ), obtained by combining data collated from the few available regional profiles with the few available national data, such as the 1998 vaccination survey [ 2 ]. Fig. 5 reports the immunity profile derived from the seroprevalence data obtained by the national survey conducted in 1996–97 (3,182 samples collected from residual sera from routine laboratory testing, in 18/20 Italian regions [ 13 ]) compared with the immunity profiles predicted by the model for the same years under the four assumptions EURO/D1, EURO/D2, UR/D1, UR/D2. Overall, the UR/D1 and the EURO/D2 assumptions match the observed profile rather well, whereas EURO/D1 and UR/D2 seem to fulfil well their expected roles of upper and lower bounds. Thus, if one disregards younger ages, where the proportion of children from 2 to 5 year of age immune to measles in 1996–97 is higher than predicted by the model (perhaps a result of increased routine coverage observed in several Italian areas since mid-1990's), the EURO FOI, as embedded in assumption EURO/D1, does not seem to perform well in the reproducing the seroprevalence profile, in contrast with UR/D1 (e.g. under EURO/D1 the same level of immunity is reached by ages 9–10 years as is reached by the data and UR/D1 by ages 17–19 years). Figure 4 The reconstructed time profile of measles routine vaccination coverage. This plausible profile of vaccination coverage was reconstructed from the very limited available national and regional data. Figure 5 Observed (ESEN) vs predicted measles immunity profiles. The serological profile from the ESEN survey is compared with model projections assuming the EURO force of infection of Edmunds et al [8] or the FOI estimated from Italian pre-vaccination case notifications but corrected for under-reporting (UR). In each case one of two assumptions is made: i) that the process of population ageing predicted for Italy has no effect upon transmission (D1) or ii) that low fertility and population ageing could lead to significant decline in transmission (D2). Discussion An attempt has been made to characterise the measles pre-vaccination epidemiological "landscape" in Italy, and also with reference to what we have referred to as the "EURO conjecture" (i.e. that age-selective under-reporting disguises underlying homology with FOIs elsewhere in Europe). There are points open to criticism in this analysis: first, the fact that by looking at "local" forces of infection, e.g. treating spatial sub-areas as autonomous epidemiological units, we disregard spatial correlations between the various local dynamics; this makes the present analysis, at best, a first step. Second, the present work relies only on case reports, the only available pre-vaccination data. Third, the increasing temporal distance between the present time and the pre-vaccination era (ending in 1976) seems to make more and more heroic the assumption that mixing patterns remain unchanged since then. Nevertheless several interesting facts emerge. Degree of effort required for eradication at the regional level Clearly, good data on immunisation coverage and reports of outbreaks of infection would provide some broad picture of patterns of susceptibility. However, as the level of vaccine-based immunity increases, the mean interval between outbreaks also increases so that case reports, even if unbiased, become much less useful as a source of timely information on patterns of susceptiblity. Also in Italy, historically, data on immunisation coverage has been very poor or in many instances completely absent; in many regions case reporting has also been very poor, forming an insensitive tool for detecting small pockets of infection, and large outbreaks, almost by definition, cannot be timely indicators of the presence of susceptibles. Hence the first motivation for the paper was to construct a map of the efforts needed for elimination of measles in Italy, which could then constitute a useful tool for prioritising regional intervention priorities in the context of the agreed WHO target for measles elimination and with the aim of avoiding such outbreaks. During the second half of the 1990's several attempts have been made in Italy to increase routine coverages, which the available data suggested had remained until then disappointingly low (in some regions there have also been campaigns targeted at older age groups, and here it should perhaps be noted that the rationale for such campaigns is re-inforced by the tendency for a lower FOI to increase the accumulation of susceptibles in older age groups which results from sub-optimal vaccination coverage). Even though surveillance data suggests incidence has reached a historic minimum during 1999–2001, national coverage was still well below 70% in 2000, with much regional heterogeneity. It is unsurprising therefore that there have been substantial epidemics causing serious concern, especially in Campania (the region with the lowest coverages) during the spring of 2002, with an estimated 20,000 cases between January and May 2002 [ 4 ]. From this standpoint the results here (i.e. basic reproductive ratios etc) suggest that the regions which seem to be the most demanding in terms of effort needed to eradicate the infection are systematically the Southern ones. This is rather problematic, as the Southern regions are those presently characterised by the lowest vaccination coverages [ 2 , 3 ]. The absolute size of the required eradication effort, as summarised by the critical coverages for routine birth vaccination, is somewhat variable and depends on the chosen mixing pattern. For Southern regions critical coverages range from 82%, under "minimally plausible" preferred mixing, to values around 95–97% under realistic assortative mixing, which represent lower and upper bounds for realistic contact patterns. Intermediate assumptions lead to intermediate figures: under "Default" mixing critical coverages for the major Southern Regions (Campania, Puglia, Sicilia) are in the range 86–88%, only two points less than the "EURO" value of 90% [ 8 ]. Keeping in mind a) that vaccination at birth is simply a "theoretical standard" (indeed critical coverages quickly increase as the age at vaccination is delayed), b) that we are assuming a 100% effective vaccine, and that, last but not least, c) the strong and persistent presence of anti-vaccination pressure groups in Italy, all these facts indicate that a substantial effort is still needed, especially in the Southern part of the country in order to approach, even in a minimal fashion, the WHO target. A major target of the Italian Measles Elimination Plan currently being initiated will be to strongly reduce regional heterogeneity in vaccine compliance. The EURO conjecture The "EURO conjecture" suggests that the measles FOI in Italy is, in fact, homologous to that observed in other European countries but that, in the case notifications data, this fact is camouflaged by a strong under reporting bias with age. Under-reporting of childhood diseases has been a major problem in Italy. It is known [ 7 , 12 ] that in the pre-vaccination era the overall reported measles incidence was an order of magnitude less than true incidence, and that the degree of under-reporting varied widely between regions. The existence of large and heterogeneous overall rates of under-reporting at the regional level does not necessarily imply, however, a bias in the (national) estimate of the force of infection: this additionally requires that i) reporting rates exhibit a significant variation with age, or that ii) spatial heterogeneity in overall reporting rates coexist with a marked spatial heterogeneity in infection patterns by age. We therefore made an extensive investigation of forces of infection at the spatial level (Regions, but also large conurbations and cities). Our feeling was that by going more deeply spatially it should have been possible to find some "footprint" of the presence of the presumed underlying EURO pattern (e.g. areas with a "high" FOI, or with significant heterogeneity in FOIs). As documented in the Results, we have not been successful in detecting such a "footprint". All "local" forces of infection are systematically substantially smaller compared to the EURO FOI, and surprisingly similar to each other in levels and shape: significant heterogeneity exists only in the youngest age groups. This raises the point, if the EURO conjecture were to be true, of what might be the social processes, if any, underlying the poor age reporting of measles cases, which are capable of so effectively camouflaging the true underlying force of infection. Indeed, in order to appear so stable over space and time, such processes should be profoundly rooted in social responses of families toward diseases of their children and/or medical practitioners' behaviour. In the absence of the relevant data any discussion of the nature of such processes must remain in the realm of speculation, but clearly there is room for bias at many points in the chain leading to reporting of a case [ 20 - 22 ](Fig. 6 ): perhaps in the historically larger families of Italy because of their size there was more direct family experience of measles and less inclination to seek medical attention when there was an uncomplicated case in the family; perhaps, with measles in young children being a normal stage of life, medical practitioners could have been less inclined to conform to the "bureaucratic" requirements of case reporting; or perhaps there could have been failures in the bureaucratic process itself. Such speculations rely on caricatures of contrasts between Italy and more northern European countries and, clearly, to provide soundly based insights a detailed study would be necessary. Nevertheless, by use of a sentinel survey selective under-reporting by age has indeed been documented in Italy by Ciofi et al [ 23 ] in recent years for varicella (still in its pre-vaccination period). However their work shows that reporting rates are somewhat higher at young ages (0–10 yr.) and subsequently tend to decrease. Therefore, assuming that current under reporting rates for varicella are in some way similar to those for measles during the pre-vaccination period, their result would not provide evidence in favour of a bias leading to underestimation of the force of infection. Figure 6 Potential sources of error in case reporting. An illustration of potential sources of failure or systematic bias in processes of case notification. We then investigated the measles incidence time series, encouraged by the fact that [ 12 ] reporting rates of measles at the regional level seemed to have remained fairly constant over time, so that (though unreliable in absolute terms) we can reasonably trust that the available incidence data should be broadly representative of the true qualitative patterns. Even in this case, however, we could not find significant footprints of an underlying "higher" force of infection. Rather the results appear to be more or less consistent with the Italian age patterns observed from case reports. Indeed the observed long-term oscillation of measles is around three years or more for most Italian regions. Such figures, though substantially different from the biennial oscillations observed for England & Wales, are by no means uncommon [ 24 ]; additionally, compared to England & Wales, they are consistent with a much higher value of the average age at infection, and more generally, with the force of infection. Though one must be cautious about these facts, it is also clear, however, that we cannot quickly dismiss them as the consequence of the presumed existence of age biases in reporting rates: it would mean that patterns of under-reporting are capable of also hiding true time patterns. Though this latter possibility can not be excluded a priori (one possibility could be that there is an association between practitioners' reporting rates and the phase of the epidemic cycle, e.g. an epidemic year vs a non-epidemic year), it appears rather unlikely in most circumstances. Indeed, if the "true" percentage age distribution of cases is broadly stationary over time, as we would expect in the pre-vaccination era (a fact that Italian data strongly suggest), then significant (and periodic) time changes in age-specific reporting rates would be needed to effectively mask true incidence patterns. Finally, our modelling work on measles in Italy, shows that the EURO assumption matches rather poorly the serological profile observed for measles in Italy in 1996/7 in the European Sero-epidemiology Network (ESEN) survey [ 8 , 25 ]. Which then was the true force of infection acting in Italy during the pre-vaccination era: the higher EURO FOI, or the lower FOI emerging from Italian case notifications data, or indeed perhaps something in between? Rather than being essentially homologous, as implicit in the EURO conjecture, the possibility that patterns of infection by measles throughout Europe could be and have been largely different (as by the way suggested for rubella by Edmunds et al . [ 26 ]) is a stimulating one. Our results provide many indications that infection patterns in Italy could indeed deviate from the "EURO" standard. However the uncertainty still present in many factors suggests that more work is urgently needed in order to better understand measles infection patterns in Italy. Be this as it may, we believe that, from a public health perspective, a quite prudent attitude is necessary: knowing that the eradication coverages computed here could just represent lower bounds of the true values, which could be substantially higher, seems to be a sufficient argument for not deviating from the target coverages suggested by WHO. Indeed, the higher R 0 values estimated for the South suggest that, even with achievement uniformly of WHO targets across the regions, elimination of measles will take longer than in the North. Conclusions The results suggest that critical vaccination coverages for elimination are likely to be higher in the south of Italy, precisely where the existing record of coverage is lowest. Substantial efforts are still required if there is to be a realistic hope of achieving WHO targets for measles elimination in Italy, particularly in the south. Notwithstanding this, the evidence does appear to suggest that the force of infection for measles in Italy is indeed somewhat lower than that applicable to other regions participating in the European Sero-Epidemiology Network. More particularly, the evidence suggests that it is probably unlikely that age biases in reporting (suggested elsewhere) could have led to an underestimate of the measles force of infection in Italy. Competing interests The author(s) declare that they have no competing interests. Authors' contributions PM conceived the work, provided the main theoretical analysis and drafted the manuscript, EMC conducted the time series analysis, JRW undertook the modelling work and SS and MCdA contributed epidemiological insight into the Italian public health context of the work. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554971.xml |
553978 | Life history traits in selfing versus outcrossing annuals: exploring the 'time-limitation' hypothesis for the fitness benefit of self-pollination | Background Most self-pollinating plants are annuals. According to the 'time-limitation' hypothesis, this association between selfing and the annual life cycle has evolved as a consequence of strong r-selection, involving severe time-limitation for completing the life cycle. Under this model, selection from frequent density-independent mortality in ephemeral habitats minimizes time to flower maturation, with selfing as a trade-off, and / or selection minimizes the time between flower maturation and ovule fertilization, in which case selfing has a direct fitness benefit. Predictions arising from this hypothesis were evaluated using phylogenetically-independent contrasts of several life history traits in predominantly selfing versus outcrossing annuals from a data base of 118 species distributed across 14 families. Data for life history traits specifically related to maturation and pollination times were obtained by monitoring the start and completion of different stages of reproductive development in a greenhouse study of selfing and outcrossing annuals from an unbiased sample of 25 species involving five pair-wise family comparisons and four pair-wise genus comparisons. Results Selfing annuals in general had significantly shorter plant heights, smaller flowers, shorter bud development times, shorter flower longevity and smaller seed sizes compared with their outcrossing annual relatives. Age at first flower did not differ significantly between selfing and outcrossing annuals. Conclusions This is the first multi-species study to report these general life-history differences between selfers and outcrossers among annuals exclusively. The results are all explained more parsimoniously by selection associated with time-limitation than by selection associated with pollinator/mate limitation. The shorter bud development time reported here for selfing annuals is predicted explicitly by the time-limitation hypothesis for the fitness benefit of selfing (and not by the alternative 'reproductive assurance' hypothesis associated with pollinator/mate limitation). Support for the time-limitation hypothesis is also evident from published surveys: whereas selfers and outcrossers are about equally represented among annual species as a whole, selfers occur in much higher frequencies among the annual species found in two of the most severely time-limited habitats where flowering plants grow – deserts and cultivated habitats. | Background Most flowering plants that are predominantly self-pollinating have an annual life history [ 1 - 3 ]. Interpretations of this association usually involve one of two main hypotheses. ( i ) Compared with perennials, annuals may generally accrue greater fitness benefits from selfing through 'reproductive assurance', i.e., because ovules may be generally more outcross-pollen-limited and/or pollen grains may be more outcross-ovule-limited [ 2 , 4 - 8 ]. ( ii ) Perennials may incur a higher fitness cost of selfing through seed discounting and inbreeding depression; hence, possibly most selfers are annuals simply because relatively few perennials can be selfers [ 9 , 10 ]. A recent third hypothesis, the 'time-limitation' hypothesis, predicts that both selfing and the annual life cycle are concurrent products of strong 'r-selection' associated with high density-independent mortality risk in ephemeral habitats with a severely limited period of time available to complete the life cycle [ 11 ]. Both the traditional reproductive assurance hypothesis and the time-limitation hypothesis involve a fitness advantage for selfing through ensuring that at least some reproduction occurs, but they involve very different selection mechanisms – pollinator/mate-limitation (where outcross pollen is not available at all due to a lack of pollinators or mates), versus time-limitation (where outcross pollen is available but arrives too late to allow sufficient time for development of viable seeds). Accordingly, these two hypotheses for selfing involve very different assumptions and predictions. The time-limitation hypothesis has direct and indirect components. The indirect component predicts higher selfing rates in annuals as a trade-off of selection for earlier reproductive maturity in annuals [ 12 , 13 ] (Figure 1a ). More rapid floral maturation is expected to result in smaller flowers with increased overlap of anther dehiscence and stigma receptivity in both space (reduced herkogamy) and time (reduced dichogamy) thus, increasing the frequency of selfing as an incidental consequence [ 12 ] (Figure 1a ). If selfing also shortens the time between flower maturation and ovule fertilization, then higher selfing rates for annuals in time-limited habitats may also be predicted as a direct fitness benefit; abbreviating the time between anthesis and ovule fertilization may ensure that there is enough remaining time in the growing season (after ovule fertilization) to allow complete seed and fruit maturation [ 11 ] (Fig 1b ). Selection favors selfing here by favoring increased overlap in anther dehiscence and stigma receptivity in both space and time, which are in turn facilitated by smaller flower size and shorter flower development time, respectively (Figure 1b ). Figure 1 Two components of the 'time-limitation' hypothesis for the evolution selfing in annuals. In (a), selfing is a trade-off of selection favoring a shorter time to reproductive maturity (fully developed flowers) under strong r-selection. As a tradeoff (dashed arrows), flowers become smaller with greater overlap in location and timing of anther dehiscence and stigma receptivity, thus increasing the rate of selfing as an incidental consequence. In (b) strong r-selection favors a shorter pollination time directly; i.e., selfing is selected for directly because it shortens the amount of time between flower maturation and ovule fertilization, thus leaving sufficient remaining time for seed and fruit maturation before the inevitable early mortality of the maternal plant under strong r-selection. In this case, smaller flower size and shorter flower development time are favored by selection because they facilitate selfing (see text). However, the two components of time-limitation cannot be separated clearly, as they operate simultaneously; i.e., earlier onset of flowering, shorter flower development time, smaller flowers and selfing can all be interpreted to have direct fitness benefits because they may all contribute directly to accelerating the life cycle [ 11 ]. Indeed, time-limitation associated with strong r-selection would be expected also to favor an acceleration of the final stage in the life cycle – seed/fruit development time (Figure 1 ) – resulting, as a trade-off, in smaller seeds and/or fruits [ 11 ]. The time-limitation hypothesis remains untested. Some recent studies have explored the rapid growth and maturation time of annuals in terms of bud development rates and ontogeny [ 13 - 15 ]. However, these studies have compared growth and development rates between selfing and outcrossing populations of only a single species. Since their effective sample size is only one, this makes it difficult to extrapolate the predominant selection pressures that may have promoted the general association of selfing with the annual life cycle. The objective of the present study was to compare, for annuals exclusively, life history traits associated with selfing versus outcrossing using several species from a wide range of plant families. Phylogenetically-independent contrasts (PIC) were used to control for confounding effects due to common ancestry among species [ 16 ]. Using a database of 118 species involving 14 families, plant size, flower size, and seed size were compared between selfing and outcrossing annuals. The time-limitation hypothesis predicts that all of these traits should be smaller in selfing annuals because the severely time-limited growing season that promotes selfing also imposes an upper limit on the maximum sizes that can be attained for plant traits [ 11 ] (Figure 1 ). The trend for outcrossers to be taller, and have larger flowers and larger seeds has often been noted [ 1 , 17 - 19 ]. We used a multi-species, across-family comparison, however, to investigate whether this trend also holds true within annuals exclusively. Data on the timing of life history stages (i.e. age at first flower, bud development time, and flower longevity) were also obtained from a greenhouse study of 25 annual species involving 5 families. The time-limitation hypothesis predicts that selfers should produce mature flowers more quickly and should have shorter flowering times. Results Data base analyses Based on phylogenetically-independent contrasts, selfing annuals had significantly shorter plant heights (Wilcoxon test for matched pairs, n = 12, T = 15.5, one-tailed P = 0.032, Figure 2a ), significantly smaller flowers (Wilcoxon test for matched pairs, n = 14, T = 13, one-tailed P = 0.0054, Figure 2b ), and significantly smaller seeds (Wilcoxon test for matched pairs, n = 13, T = 13, one-tailed P < 0.01, Figure 2c ). Figure 2 (a) Plant height contrasts for 13 selfing and outcrossing pairs (some points overlap), where each pair consists of the median value of the selfing and outcrossing species within one family. (b) Flower size contrasts for 14 selfing and outcrossing pairs. (c) Seed size constrasts between 14 selfing and outcrossing pairs. (See Appendix A for list of families and species). Greenhouse study Bud development time (Figure 3 ) and flower longevity (Figure 4 ) were significantly (P < 0.05) shorter in selfing annuals in all of the families except the Fabaceae (P = 0.123 and P = 0.056 respectively). Selfing annuals also had significantly shorter bud development times (Figure 5 ), and floral longevities (Figure 6 ) in three of the four genus pairs. Selfing and outcrossing annuals of the genus Ipomoea did not differ significantly in either bud development time (P = 0.402) or flower longevity (P = 0.328). Age at first flower was not significantly related to mating system for any of the family or genus comparisons (P > 0.05; data not shown). Figure 3 Mean (SE) bud development time for selfing and outcrossing species within each of 5 families. For each species, n = 4 or 5. P – values are from ANOVA. (See Appendix B for species list). Figure 4 Mean (SE) flower longevity for selfing and outcrossing species within each of 5 families. For each species, n = 4 or 5. P – values are from ANOVA. (See Appendix B for species list). Figure 5 Mean (SE) bud development time for selfing and outcrossing species within each of 4 genus pairs. For each species, n = 4 or 5. P – values are from ANOVA. (See Appendix B for species list). Figure 6 Mean (SE) flower longevity for selfing and outcrossing species within each of 4 genus pairs. For each species, n = 4 or 5. P – values are from ANOVA. (See Appendix B for species list). Discussion There is a rich body of theory and empirical work on the evolution of selfing in flowering plants [e.g. [ 1 , 2 , 4 - 7 , 9 , 10 ]], but practically none of it involves an explicit role of selection involving time-limitation. The present paper is only the second to explore the implications of the time-limitation hypothesis and contribute to the maturation of this idea. According to the time-limitation hypothesis, selfing in annuals has evolved as a consequence of strong r-selection in ephemeral habitats, resulting either as an indirect consequence (trade-off) of selection for shorter time to reproductive maturity (Figure 1a ), or as a direct consequence of selection for shorter pollination time, i.e., the time between flower maturation and ovule fertilization (Figure 1b ), or both [ 11 ]. Consistent with the predictions of this hypothesis, we found, using phylogenetically-independent contrasts, that (compared with outcrossing annuals) selfing annuals in general had significantly shorter plant heights, smaller flowers, shorter bud development time, shorter flower longevity and smaller seed sizes. At the same time, these results are not inconsistent with the predictions of selection resulting from pollinator/mate-limitation associated with the traditional reproductive assurance hypothesis. Just as with many situations where two different mechanisms can potentially produce the same outcome/pattern, it is not easy here to clearly distinguish between the roles of "pollinator/mate-limitation" and "time-limitation". Nevertheless there are two important contributions from our study: First, in reporting significant life history differences between selfers and outcrossers, our multi-species study is unique in its comparison of monocarpic annual species exclusively. All previous multi-species studies of trait comparisons between selfers and outcrossers have involved variable mixes of monocarpic and longer-lived polycarpic species. Second, by comparing annuals exclusively, our results provide indirect support for the time-limitation hypothesis, not by rejecting the role of pollinator/mate-limitation, but rather by representing a system in which it is more plausible to argue for the role of time-limitation; i.e., compared with pollinator/mate-limitation, time-limitation as a selection factor favoring selfing is likely to have been much stronger, more persistent and more widespread. The strength of this argument lies in the fact that the annual life history is unequivocally a product of some type of time-limitation favoring an abbreviated life cycle, which is promoted by (among other things) selfing (as opposed to outcrossing) (Fig. 1 ). It is much less plausible to suspect that selection associated with pollinator/mate-limitation has been sufficiently strong and persistent to favor selfing in such a wide range of annual taxa across the many genera and families considered here. We emphasize, therefore, that for annuals the time-limitation hypothesis provides a more parsimonious explanation for the differences in traits between selfers and outcrossers. We consider each of these traits in turn below. Plant height and time to anthesis Taller plants may attract more pollinators and, hence, experience greater outcrossing rates [ 20 , 21 ]. The pollination benefit of being relatively tall, therefore, is presumably experienced only by outcrossers. If, however, selfers have evolved from outcrossers [ 3 ], then why should selfers be shorter than their outcrossing ancestors? The relatively small size, including short height of selfers can be predicted as an indirect consequence of selection, from time-limitation, favoring precocious maturation time [ 22 , 23 ] (Figure 1a ). In the present study, however, selfers and outcrossers did not differ significantly in age at first flower. Andersson [ 18 ] found similar results between selfing and outcrossing populations of Crepis tectorum . Arroyo [ 24 ], however, reported that selfing individuals of Limnanthes floccosa flowered earlier than the outcrossing L. alba , as predicted by the time-limitation hypothesis. The results for flowering times in the present study may be confounded by the controlled greenhouse environment of constant day-length, temperature and moisture regime. In the field, flowering times may be triggered by environmental cues. L. floccosa , for example, uses soil moisture to trigger the early onset of flowering, thus escaping the detrimental effects of soil desiccation during seed development [ 24 ]. Note also that age at first flower is only a crude estimate of time to reproductive maturity. Future studies may employ more detailed measures such as rate of mature flower production. Flower size One of the most well established trends of predominantly self-fertilizing species is their reduced flower sizes compared with outcrossing species [ 1 , 17 ]. The present results indicate that this trend is also evident even within annuals exclusively. In all but three of the 14 PICs, selfing annuals had smaller flowers than the outcrossing annuals (Figure 2b ). Outcrossers and selfers had similar flower sizes in the Fabaceae and Plantaginaceae. In the Poaceae, outcrossing annuals had smaller flowers than selfers. Under the time-limitation hypothesis, smaller flowers and selfing may be tradeoffs of selection for precocity (Figure 1a ), or smaller flowers may be favored by selection because they promote selfing and hence, direct fitness benefits by abbreviating pollination time (Figure 1b ). Also, if selfing evolves from outcrossing (by whatever mechanism), then selection may subsequently favour a reduction in flower size since relatively large flowers are no longer needed to attract pollinators. Hence, higher fitness may result if the resources required to construct and support these larger flowers are invested instead in other functions (e.g. seed and fruit development) [ 17 ]. Bud development time Selfers had significantly shorter bud development times in all but one of the independent family contrasts (Figure 3 ) and all but one of the genus comparisons (Figure 5 ). Results from previous studies, however, are inconsistent. Shorter bud development times were found in selfing populations of Mimulus guttatus [ 25 ] and in Clarkia xantiana [ 14 ]. However, no significant differences in bud growth rates were found between the selfing and outcrossing populations of C. tembloriensis [ 15 ]. Hill, Lord and Shaw [ 13 ] reported that flowers from selfing populations of Arenaria uniflora develop over a longer period of time than observed in outcrossing populations. In the field, selfing populations of A. uniflora were also observed to flower at the same time or even later than outcrossing populations [ 13 ], suggesting that time-limitation is not currently a strong selection pressure. Self-fertilization in A. uniflora may have arisen through reproductive assurance in response to competition for pollinators [ 7 ]. The evolution of self-fertilizing species from outcrossing progenitors has occurred repeatedly and independently in several lineages [ 1 , 3 , 14 ], each of which may have been associated with different contexts of natural selection vis-à-vis the fitness benefits of selfing. Flower longevity The families and genera in which selfers had shorter bud development times also had significantly shorter flower longevities (Figure 4 ). In fact, all of the selfers had flowers that remained open for less than four days (except in Trifolium hirtum ; Fabaceae), with a large proportion of flowers open for only one day, which is common amongst self-fertilizing species [ 17 ]. The present data again indicate that this generalization apparently holds true even within annuals exclusively. By having flowers that remain open longer, outcrossers increase the probability of visitation by pollinators and successful cross-pollination [ 17 ]. This fitness benefit is realized, however, only if there is sufficient time remaining after cross-pollination to complete seed and fruit development before the maternal plant succumbs to density-independent mortality in strongly r-selecting habitats [ 11 ]. If time is limiting in this context, selection should favor selfing (Figure 1b ) with no advantage in having long-lived flowers. It is important to note that our data measure maximum flower longevity, since there were no pollinators in the greenhouse, nor was hand pollination conducted. Pollination has been shown to induce floral senescence in numerous species [ 26 ]. This effect was not tested on any of the study species, which means that our observed flower longevities in outcrossing species may be longer than would normally be seen in the wild. Nevertheless, since selfing may have evolved as a method of shortening pollination time, and flower longevity was used as a measure of pollination time, the maximum floral longevity gives an indication of how long outcrossers can delay flower abscission or self-pollination (i.e. through delayed selfing). Seed size Strong r-selection associated with the annual life form presumably favors wide dispersal mechanisms (for colonizing new and distant sites) which may be conferred by small seed sizes [ 19 ]. The reproductive assurance hypothesis would predict, therefore, that most selfers are annuals because annuals are more likely than perennials to disperse further, or colonize new habitats where conditions are unsuitable for successful outcrossing (because of a shortage of mates or pollinators) and where selfing, therefore, provides reproductive assurance. The present study indicates that even among annuals only, selfers have smaller seeds than outcrossers (Figure 2c ). Future studies are required to test whether smaller-seeded selfing annuals are more likely than their outcrossing annual relatives to disperse further or colonize new habitats and thereby incur potential reproductive assurance benefits of selfing. An alternative explanation, however, is offered by an extension of the time-limitation hypothesis: strong r-selection favors an acceleration of all stages of the life cycle (Figure 1 ), including not only earlier reproductive maturity (Figure 1a ) and a shorter pollination time (facilitated through selfing) (Figure 1b ), but also a shorter seed and fruit maturation time, which, on a per-seed basis, is facilitated in turn through the production of smaller seeds. Andersson [ 18 ] found that self-fertilizing individuals of Crepis tectorum took an average of 16 days for fruit maturation, whereas outcrossing individuals of the same species required 43.3 days. Small seed size may also be simply a trade-off of selection for high fecundity, also favored by strong r-selection [ 11 ]. Habitat selection and time-limitation While most selfers are annuals, it is not the case that most annuals are selfers. An unbiased literature survey [ 27 ] suggests that roughly half of all annual species are selfers and half are outcrossers. If, however, selfing annuals evolved in habitats with a short window of time for completing the life cycle (Figure 1 ), then selfing annuals should be significantly more common than expected (i.e. comprising greater than 50% of resident annuals) within habitats associated with historically regular, early-season disturbances (e.g. cultivated fields, gardens), or in habitats where severe droughts follow quickly after a wet season (i.e. deserts, Mediterranean climates, vernal pools). Hence, we should expect to find more selfers than outcrossers among annual weeds of cultivated habitats and among desert annuals in particular. Similarly, for annuals with both selfing and outcrossing ecotypes or races, we should expect selfers (or a higher selfing rate) to be more commonly associated with these severely time-limited habitats [ 11 ]. While rigorous tests of these predictions have yet to be explored, some preliminary support is available from published surveys. From a representative sample of Mediterranean annuals [ 28 ], we find a much greater representation of selfers: i.e. 34 selfers versus 11 outcrossers. Selfing and outcrossing desert annuals have been shown to be distributed along a moisture gradient. Outcrossing annuals are found generally in the wetter areas and selfers in the more arid zones, as seen in Clarkia xantiana [ 29 ] and between outcrossing populations of Limnanthes alba and its selfing relative L. floccosa [ 24 ]. Since the length of the growing season is limited by the amount of moisture in the soil, selfers have a much narrower window of time to complete their life cycle before desiccation. During a severe drought, seed production in L. alba was reduced by one sixth, whereas the seed set of L. floccosa found in the same area was virtually unaffected by the identical drought [ 24 ]. The association between 'weediness' and self-fertilization has also been noted [ 2 , 30 ]. An extensive survey of colonizing herbaceous plants of Canada showed that agricultural weeds of row crops and grain fields are almost exclusively annuals, and most of these are self-compatible [ 31 ]. A published list of the world's worst weeds of agricultural crops [ 32 ] includes 76 species, 41 of which are annuals. Based on previous literature, we were able to identify the breeding system for 24 of these annuals, and, as predicted, the majority (20 out of 24) are selfers. Conclusions Botanists have long known that selfing is particularly associated with the annual life cycle in flowering plants [ 2 ]. The present study shows further that, among annuals exclusively, selfing is particularly associated with shorter plant heights, smaller flowers, shorter bud development time, shorter flower longevity and smaller seed sizes compared with annuals that are outcrossing. Also, in spite of the null prediction that selfing and outcrossing annuals should be equally represented if there is no bias associated with time-limitation, we found instead that two of the most time-limited habitats on earth that support flowering plants have a significantly higher percentage of selfers among the resident species that are annuals. Because we focused on annual species only, all of these results are explained more parsimoniously by selection associated with time-limitation than by selection associated with pollinator/mate limitation. The role of pollinator/mate-limitation (as traditionally associated with the reproductive assurance hypothesis for the evolution of selfing) is likely to be of greater importance in longer-lived polycarpic species (not considered here), simply because by comparison, there is no convincing basis to argue that selection associated with time-limitation is likely to have been important in species with longer life cycles. We suggest therefore, that most selfers, because most of them are annuals, are likely to have evolved not because of fitness benefits through reproductive assurance associated with selection from pollinator/mate limitation, but rather because of fitness benefits associated with selection from time limitation. The effect of time-limitation under strong r-selection is to minimize the duration of the life cycle, with selfing favored directly (Figure 1b ) and/or indirectly (Figure 1a ). There is no basis for predicting that either mechanism is more probable than the other; both are likely to operate simultaneously and perhaps indistinguishably. Indeed, the predicted effects under direct and indirect selection involve the same phenotypic outcome for the same suite of traits (Figure 1 ). It is particularly significant that the shorter bud development time reported here for selfing annuals is predicted explicitly by the time-limitation hypothesis but not by selection associated with pollinator/mate limitation. Although, we cannot of course rule out the possibility that shorter bud development time may be a pleiotropic consequence of the evolution of autonomous selfing through other mechanisms. Designing empirical studies that clearly distinguish between mechanisms involving time-limitation versus pollinator/mate limitation remain a challenge but we anticipate that our results and our discussion of these issues may help to inspire further research along these lines. Future studies may be designed to test more directly the role of limited pollination time ( vis-à-vis Figure 1b ) by comparing the time required for effective pollination under selfing versus outcrossing for closely related species or ecotypes within natural habitats, taking care of course to control for other aspects of the pollination environment (such as mate and pollinator availability) that might affect time-to-effective pollination. Methods Data base analyses The literature was surveyed to obtain breeding information (i.e. selfing versus outcrossing) for as many annuals species as possible. For each species, data on plant height, flower size, and seed size were obtained where possible from standard floras and other published literature. A complete database was assembled for 118 species from both Europe and North America, involving 14 families (Table 1 ). For each species, the maximum published value for each trait was used. Plant height was the maximum recorded vertical extent of the plant. The measure used for flower size depended on the usual convention specific for each family, e.g. maximum petal length, corolla width, lemma length (in the Poaceae). Seed size was measured as the length of the longest axis. Within each family, for each trait, the median value across selfing species and the median value across outcrossing species was calculated and used in the phylogenetically-independent contrasts. Table 1 Species list, with breeding system (O – outcrosser; S – selfer), for database from published literature. Family Family Species Species Asteraceae Malvaceae Anthemis cotula (O) Abutilon theophrasti (S) Cosmos bipinnatus (O) Hibiscus trionum (O) Centaurea cyanus (O) Malva neglecta (O) Centaurea montana (O) Malva rotundiflora (S) Crepis capillaris (O) Crepis tectorum (O) Plantaginaceae Helianthus annuus (O) Plantago arenaria (O) Lapsana communis (O) Plantago virginica (S) Matricaria maritima (O) Matricaria matricarioides (S) Poaceae Senecio viscosus (S) Aira praecox (O) Senecio vulgaris (S) Avena fatua (S) Silybium marianum (S) Avena sativa (S) Sonchus oleraceus (S) Bromus hordeaceus (S) Xanthium strumarium (S) Bromus secalinus (O) Bromus sterilis (S) Boraginaceae Bromus tectorum (S) Anchusa arvensis (O) Desmazeria rigida (S) Borago officinalis (O) Echinochloa crus-galli (S) Lappula squarrosa (S) Hordeum vulgare (S) Myosotis arvensis (S) Lolium temulentum (S) Myosotis ramosissima (S) Panicum miliaceum (S) Myosotis stricta (S) Phalaris canariensis (O) Plagiobothrys calandrinioides (S) Poa annua (S) Secale cereale (O) Brassicaceae Setaria italica (S) Arabidopsis thaliana (S) Setaria verticillata (S) Berteroa incana (O) Setaria virdis (S) Brassica juncea (O) Triticum aestivum (S) Brassica nigra (O) Zea mays (O) Brassica rapa (O) Cakile edentula (S) Polemoniaceae Capsella bursa-pastoris (S) Allophyllum gilioides (S) Cardamine hirsute (S) Allophyllum integrifolium (S) Descurainia pinnata (S) Collomia grandiflora (O) Diplotaxis muralis (O) Collomia linearis (S) Erucastrum gallicum (S) Gilia australis (S) Erysimum cheiranthoides (O) Gilia capitata (O) Erysimum repandum (S) Gilia caruifolia (O) Lepidium sativum (O) Gilia clivorum (S) Lepidium campestre (S) Gilia inconspicua (S) Lepidum ruderale (S) Gilia millefoliata (S) Rorippa palustris (S) Gilia sinuata (S) Sinapis alba (O) Gilia tenuiflora (O) Sinapis arvensis (O) Gilia transmontana (S) Sisymbrium officinale (S) Gilia tricolor (O) Thlaspi arvensis (S) Navarretia atrictyloides (O) Thlaspi perfoliatum (S) Navarretia squarrosa (S) Caryophyllaceae Polygonaceae Agrostemma githago (O) Fagopyrum esculentum (O) Arenaria serpyllifolia (S) Polygonum aviculare (S) Cerastium nutans (O) Polygonum convolvulus (S) Silene dichotoma (O) Polygonum hydropiper (S) Silene noctiflora (S) Polygonum lapathifolium (S) Spergula arvensis (S) Polygonum persicaria (S) Stellaria media (S) Ranunculaceae Fabaceae Myosurus minimus (S) Medicago lupulina (O) Ranunculus reptans (O) Trifolium arvense (S) Ranunculus sceleratus (O) Trifolium aureum (S) Trifolium campestre (S) Scrophulariaceae Vicia sativa (S) Chaenorrhinum minus (S) Vicia tetrasperma (O) Veronica agrestis (O) Veronica arvensis (S) Lamiaceae Veronica peregrina (S) Galeopsis tetrahit (O) Veronica persica (S) Lamium amplesicaule (S) Lamium purpureum (S) Apiaceae Aethusa cynapium (S) Anethum graveolens (O) The contrasts were based on 14 phylogenetically-independent pairs, where each pair consisted of median values of the selfing and outcrossing species within one family, which by definition are species that are more closely related to each other than to any other species in the data set [ 19 ]. For plant height, only 13 pairs were used due to missing information. The data were analyzed using a Wilcoxon matched pairs test. Greenhouse study The species included in this study were chosen if there was a known breeding system, if germinable seeds were available, and if a complementary species (i.e. in the same family with the opposite breeding system) was known and could also be obtained as germinable seeds. Seeds were obtained from a variety of sources; Herbiseed, Rancho Santa Ana Botanic Gardens, Chiltern Seeds, S&S Seeds, and the National Plant Germplasm System. Our search lead to an unbiased sample of 25 candidate species, allowing five pair-wise family comparisons and four pair-wise genus comparisons (Table 2 ). Table 2 List of species, with breeding system (O – outcrosser; S – selfer), used in the greenhouse study
. Family Family Species Species Asteraceae Convolvulaceae Crepis capillaris (O) Ipomoea hederacea (S) Helianthus annuus (O) Ipomoea purpurea (O) Matricaria maritime (O) Matricaria matricarioides (S) Fabaceae Senecio viscosus (S) Lupinus bicolor (O) Senecio vulgaris (S) Lupinus nanus (S) Lupinus succulentus (O) Boraginaceae Trifolium hirtum (S) Borago officinalis (O) Myosotis arvensis (S) Lythraceae Cuphea laminuligera (O) Brassicaceae Cuphea lanceolata (O) Brassica juncea (O) Cuphea lutea (S) Brassica nigra (O) Capsella bursa-pastoris (S) Polemoniaceae Cardamine hirsuta (S) Navarretia squarrosa (S) Sinapis alba (O) Phlox drummondii (O) Sinapis arvensis (O) Most species were germinated in 15 cm pots filled to 3 cm below the top with standard potting soil (Promix BX © ). Pots were placed in a greenhouse and watered daily until the appearance of their first true leaves. Subsequently, they were watered uniformly every second day to ensure that the soil was kept moist. Some species were germinated in a petri-dish in a growth chamber (23°C, 12 hour cycles of light and dark), after which they were transplanted into pots and placed in the greenhouse. Each species was replicated five times, with one plant per pot. Pots were arranged randomly on benches at a density of 1 pot per 0.093 m 2 . The plants were exposed to 16 hours of daylight each day, with maximal natural light levels at ca 1200 μE. Before sunrise and after sunset, artificial lights (250–300 μE) were used to supplement the light exposure to 16 hours of light per day. The greenhouse was kept at an average temperature of 23.1°C during the day and dropped to 20.0°C at night. The plants were fertilized every 2 weeks with 200 ml per pot of a 2g/L concentration of 20–20–20 N-P-K fertilizer. For each plant, age at first flower, bud development time, and flower longevity were measured. Emergence of the first pair of true leaves, after the cotyledons, was considered day 1 of the plant's life. Age at first flower was measured in days from day 1 to when the first flower opened on each plant. Bud development time (n = 3 buds per plant) was calculated as the number of days from the first appearance of a new bud until the flower opened. The same three buds on each plant were then monitored every day after opening, and the number of days until the flower senesced (flower longevity) was recorded for each. A flower was considered to be senesced when the corolla wilted, fell apart, or became discolored, as designated by Primack [ 17 ]. Any flower that was open for only one day was considered a one-day flower, regardless of whether it was open for the whole day or only part. Flowers in the Asteraceae were considered withered when the whole inflorescence had senesced, rather than the individual florets [ 17 ]. For bud development time and flower longevity, the three replicate measurements for each plant were averaged, and then these values for the five replicate plants were averaged to obtain a mean value for the species. The data were analyzed with a standard least squares one-way analysis of variance (ANOVA) model, with a post-hoc contrast between selfers and outcrossers. These analyses were done for each family and genus separately in order to control for phylogeny at these levels. In cases where the data were non-normal, a log-transformation was applied which corrected the distribution. Authors' contributions RS collected the data, performed most of the analyses, participated in the design of the study, and wrote the first draft as a B.Sc.(Hons) thesis. LWA conceived of the study, participated in its design and coordination, and wrote the final draft for submission to BMC. Both authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553978.xml |
553987 | Molecular strategies to inhibit HIV-1 replication | The human immunodeficiency virus type 1 (HIV-1) is the primary cause of the acquired immunodeficiency syndrome (AIDS), which is a slow, progressive and degenerative disease of the human immune system. The pathogenesis of HIV-1 is complex and characterized by the interplay of both viral and host factors. An intense global research effort into understanding the individual steps of the viral replication cycle and the dynamics during an infection has inspired researchers in the development of a wide spectrum of antiviral strategies. Practically every stage in the viral life cycle and every viral gene product is a potential target. In addition, several strategies are targeting host proteins that play an essential role in the viral life cycle. This review summarizes the main genetic approaches taken in such antiviral strategies. | Introduction HIV-1 is a lentivirus belonging to the retrovirus family. The virus is diploid and contains two plus-stranded RNA copies of its genome. The approximately 9 kb RNA genome encodes at least 9 proteins, Gag, Pol, Env, Tat, Rev, Nef, Vif, Vpu and Vpr of which only the former five are essential for viral replication in vitro. HIV-1 primarily infects CD4 + T-lymphocytes and monocytes/macrophages, but also astrocytes and cells of the central nervous system (brain microglial cells) are targets. The infection spreads to the lymphatic tissue that contains follicular dendritic cells that may act as a storage place for latent viruses. Over time, virus replication leads to a slow and progressive destruction of the immune system. The development of possible methods that can delay progression of the infection or block replication of HIV-1 in infected individuals has been the subject of dedicated research efforts over the past decades. One important issue is that HIV-1 makes use of the replication machinery of the host cell, which minimizes the number of potential viral targets. On the other hand, the close host-virus relationship limits the evolutionary freedom for the viral components that interact with the host molecules. The aim of this review is to take a comprehensive look at the molecular, intracellularly based antiviral strategies that have been reported in literature, and to discuss their potential for development into clinical protocols. We will not discuss vaccine-based strategies that recently have been reviewed in [ 1 ] and [ 2 ]. Interfering strategies against HIV-1 The inhibition strategies can be divided into two groups: The RNA-based strategies including anti-sense RNA (or other chemically modified nucleic acids), RNA decoys (sense RNA), ribozymes, RNA aptamers, small interfering RNA (siRNA), microRNAs (miRNAs) and the protein-based strategies including transdominant negative proteins (TNPs), chimeric proteins (fusion proteins), nucleases, anti-infective cellular proteins, intracellular single-chain antibodies (sFvs) and monoclonal antibodies (Mabs). In addition, other strategies based on suicide genes, protease inhibitors and nucleoside or non-nucleoside analogues have shown to possess the ability to reduce HIV-1 replication. The HIV-1 life cycle including the inhibiting strategies targeted against the various steps in the viral life cycle is summarized in Fig. 1 and listed in table 1 . Below follows a more detailed description of the strategies taken to target individual steps of the viral life cycle. Note that strategies targeting the viral genes or mRNA directly all possess an uncertainty as to what viral function(s) are affected due to the overlapping nature of some of the reading frames [ 3 ]. Figure 1 Summarization of the HIV-1 life cycle and the inhibiting strategies targeting the different steps in the viral life cycle. Table 1 Interfering strategy Target RNA/protein Interference site(s) Mechanism References Anti-sense RNA Cellular CCR5 and CXCR4 co-receptors Viral entry Inhibition of CCR5 and CXCR4 gene expression 6, 18, 19 Psi-gag and U3-5'UTR-gag-env regions Pre-integration Co-packaged with genomic RNA, inhibits RT in incoming virions 6 Cellular CyPA gene Pre-integration The skipping of internal CyPA encoding exons reduces CyPA biosynthesis and thereby inhibits the reverse transcription 37 Tat/TAR interaction HIV-1 transcription Inhibits transcriptional regulation of HIV-1 gene expression 6, 7, 45, 76 Rev/RRE interaction Nuclear export Inhibits transport of unspliced and single spiced viral RNAs 6 5'UTR HIV-1 translation Inhibits the translation process 6 Psi-gag region Viral assembly Inhibits packaging of genomic RNA 6, 7, 45 5'-leader-gag region Viral assembly Inhibits the formation of Gag and Env multimeric complexes during viral assembly. 7, 18 Env and Vif encoding regions Viral assembly Inhibits env and vif gene expression 70 Nef encoding region Viral release Inhibits nef gene expression and thereby CD4 and MHC I downregulation 7 Pol encoding region Viral maturation Inhibits pol gene expression 70 RNA decoys RT enzyme Pre-integration Competes with HIV-1 RNA for the binding of RT 6 HIV-1 TAR region Pre-integration Competes with cellular tRNA 3 Lys for the binding to RT and primes the reverse transcription from the TAR region instead of the PBS region 6 Tat and Tat-containing RNA polymerase II transcription complexes HIV-1 transcription Inhibits Tat regulated transcription 6, 7, 18, 51 Rev protein Nuclear export Recruits Rev molecules and thereby prevents their interaction with the viral transcript 6 NC domain of the Gag protein Viral assembly Inhibits packaging by interfering with the NC domains ability to recognize the genomic RNA 6, 45 Ribozymes Cellular CCR5 and CXCR4 co-receptors Viral entry Cleaves CCR5 and CXCR4 mRNAs 6, 18 HIV-1 Gag and Pol encoding region and the U5 region Pre-integration Cleaves the viral RNA before reverse transcription is completed 6, 36 RRE and the Rev encoding region Nuclear export Cleaves the viral RNA 6, 7 U5 HIV-1 translation Cleaves off the 5'-cap structure localized on HIV-1 mRNAs 6, 7 Psi Viral assembly Cleaves HIV-1 RNAs before packaging 6, 7 Gag encoding transcripts Viral assembly Inhibits the formation of multimeric Gag and Env complexes 7, 18 SU encoding region Viral assembly Cleaves different conserved regions in the SU sequence 7 Nef encoding region Viral release Inhibits downregulation of CD4 and MHC I 7 RNA aptamers RT enzyme Pre-integration Displays high affinity and specificity for the RT enzyme and acts as templates analogues 31 Rev protein Nuclear export Possesses higher affinity for Rev than the RRE sequence and can therefore interfere with Rev function 57 siRNA Cellular CCR5 and CXCR4 co-receptors Viral entry Impairs the SU-chemokine co-receptor interaction 21, 22 CD4 protein Viral entry CD4 protein expression inhibited 23, 24 CD4-binding domain of the SU protein Viral entry Inhibits the CD4-SU interaction 26 The viral LTR region or the vif and nef encoding regions Pre-integration Guides the viral genomic RNA towards a siRNA-mediated destruction 34, 52 RT encoding region Pre-integration Inhibits RT gene expression 35 Cellular CyPA gene Pre-integration Reduces CyPA biosynthesis and thereby the reverse transcription 37 CA encoding region Pre-integration Mediates cleavage of pre-spliced viral RNA in the cytoplasm and prevents integration 23, 24, 42 Tat encoding region HIV-1 transcription Inhibits Tat transactivation 35, 49, 50 NF-κB p65 subunit HIV-1 transcription Inhibits NF-κB transcriptional activation 35, 49 3'-terminus of the nef gene HIV-1 transcription Mediates cleavage of all spliced and unspliced RNA produced from the provirus 42 Rev transcript Nuclear export Inhibits Rev mediated export of unspliced and single spliced RNAs 49, 61 Gag and Nef encoding regions HIV-1 translation Mediates cleavage of both spliced and unspliced RNA produced from the provirus 23, 24, 34, 42 shRNA/ miRNA Nef encoding region HIV-1 translation nef shRNAs act by blocking RNA stability or RNA translation 62 Transdominant negative proteins (TNPs) Interactions between Tat/TAR complex and cellular co-factors HIV-1 transcription Tat-mutants inhibit the function of the Tat protein by recruiting important cellular co-factors 7, 18, 45 Rev protein Nuclear export Rev-mutants e.g. act by preventing the interaction with cellular co-factors or by sequestering the Rev protein in the cytoplasm 7, 18, 25, 57, 58, 59 Cellular Sam68 Nuclear export Sam68 mutants inhibit Sam68 transactivation of RRE and Rev function 60 Cellular Tsg101 Viral assembly Tsg101 mutants inhibit the transport of the Gag polyprotein into multivesicular bodies 71 Vif protein Viral assembly Vif mutants block an early processing of the Gag protein 66 Cellular INI1 Viral assembly INI1 mutants e.g. interact with the integrase domain of the Gag-Pol polyprotein and interfere with prober multimerization of Gag and Gag-Pol 39 The formation of Gag and Env multimeric complexes Viral assembly E.g. interferes with complex formation 4, 6, 18 Nef protein Viral release Nef mutants e.g. inhibit CD4 downregulation 66 SU protein Viral release Overexpressed CD4 variants bind and sequester virion progeny within the cell 19 HIV-1 protease Viral maturation Pro-mutants prevent protease activation 7 Chimeric / fusion proteins SU protein Viral entry A tetrameric version of sCD4, PRO542, which is fused to the conserved region of IgG2, prevents the CD4-SU interaction 8, 13 Proviral DNA Pre-integration An IN targeted sFv-nuclease fusion protein associates with the pre-integration complex and cleaves proviral DNA after integration has occurred 7, 18 TAR element HIV-1 transcription Designed Tat-nuclease fusion proteins recognize and cleave all HIV-1 RNA transcripts 5 RRE sequence Nuclear export Designed Rev-nuclease fusion proteins recognize and cleave all HIV-1 RNAs carrying the RRE sequence 5 Rev protein Nuclear export A NS1RM-Rev mutant, with a dominant retention activity, forms mixed oligomers together with Rev and inhibits nuclear export 7, 57 The TAR and RRE elements HIV-1 transcription / nuclear export A designed fusion protein, Tev, containing the RNA binding domains of both Tat and Rev fused to a nuclease, inhibits both early and late viral gene products 5 Viral genomic RNAs Viral assembly Gag-, Vpr- and Nef-nuclease fusion proteins cleaves viral RNA, either during or after the viral assembly 5, 7 Psi-element Viral assembly A NC-nuclease fusion protein recognizes and cleaves all unspliced RNAs in the cytoplasm 5 HIV-1 protease Viral maturation An overexpressed Vpr fused to several protease cleavage sites overwhelms the protease activity by a competitive mechanism 7, 74 Nucleases Tat encoding region HIV-1 transcription Inhibits Tat transactivation 6, 7, 45 TAR element HIV-1 transcription Inhibits Tat transactivation 6, 7, 45 Chemokine ligands Cellular CCR5 and CXCR4 co-receptors Viral entry E.g. interacts directly with the co-receptors, mediates receptor blockade or mediates receptor down-regulation 8, 9, 11, 12, 13, 14, 16 Anti-infectious cellular proteins SU protein Viral entry A truncated form of CD4, sCD4, inhibits the fusion event by binding to the SU protein and thereby extending the distance to the TM protein 8, 13, 19 Intracellular antibodies (sFvs) SU protein Viral entry Inhibits the CD4-SU interaction 18 The TM pre-hairpin intermediate Viral entry Inhibits the interaction between the fusion peptide and the cell membrane 29 RT enzyme Pre-integration Inhibits RT function 7, 18 IN enzyme Pre-integration Inhibits IN function 7, 18 Tat protein HIV-1 transcription Interacts with the Tat protein and restrains it in the cytoplasm 7, 18 Rev protein Nuclear export Recruits Rev in the cytoplasm 7, 18, 25, 57 The CD4 binding region of the SU protein Viral assembly Interacts with the Env protein and restrains it in the ER 7, 18 Monoclonal antibodies (Mabs) Cellular CCR5 and CXCR4 co-receptors Viral entry E.g. inhibit the SU-chemokine co-receptor interaction, HIV-1 fusion or entry 12 Extracellular loop on CCR5 SU-chemokine co-receptor interaction Inhibits HIV-1 fusion and entry 12 Nucleoside analogues (NRTIs) RT enzyme Pre-integration Prevents the continued polymerization of the DNA chain 8 Non-nucleoside analogues (NNRTIs) RT enzyme Pre-integration Interact directly and non-competitively with the RT enzyme and inhibits its function 8 Integrase inhibitors (Oligonucleotides, dinucleotides and chemical agents) IN enzyme Pre-integration These inhibiting agents either block the catalytic function of the IN enzyme by binding to the integrase binding site located in the viral DNA, or by interacting with the catalytic core domain of the IN enzyme itself 40, 41 Protease inhibitors Protease enzyme Viral maturation Act as transition state analogous and bind to the protease more tightly than the natural substrate 11, 8, 73 Examples of other inhibiting agents Cellular CCR5 and CXCR4 co-receptor Viral entry Chemokine ligands potently inhibit the SU-chemokine co-receptor interaction 8, 9, 10, 11, 12, 13 Cellular CCR5 and CXCR4 co-receptors Viral entry Designed peptides e.g. act by disrupting helix-helix interactions, which may influence co-receptor structure, or by associating with the co-receptor surfaces and thereby inhibit the interaction with the SU protein 8, 12 Cellular CXCR4 co-receptor Viral entry AMD3100, a small organic molecule, acts by spanning the main ligand-binding cavity of CXCR4, which constrains the co-receptor in an inactive conformation 12 Cellular CCR5 co-receptor Viral entry Cyclophilin-18, a protein derived from T. Gondii acts as a CCR5 antagonist and thereby inhibits fusion and infectivity of R5 HIV-1 isolates 17 SU protein Viral entry CV-N, a 11 kDa protein with high affinity for the SU protein, inhibits the SU-CD4 interaction 15 The N- and C-peptide regions on the TM pre-hairpin intermediate Viral entry Designed N-, C-, and D-peptides interacts with the pre-hairpin intermediate and inhibit the fusion event 13, 27, 28 RT enzyme Pre-integration Small peptides, about 15–19 amino acid long, act by interfering the dimerization process of the RT enzyme 30 The Tat/TAR interaction HIV-1 transcription The TR87 compound acts by competing with Tat for binding to TAR-RNA 46 Protein /TAR RNA interaction HIV-1 transcription Pyrrolo [2,1-c][1,4]benzodiazepine-oligopyrrolo hybrids act by interrupting binding of cellular proteins and Tat to the TAR-RNA 47 Protein /TAR RNA interaction HIV-1 transcription Aromatic polyamidines carrying a Br atom inhibit cellular and viral protein-TAR RNA interactions 48 Cellular NF-κB HIV-1 transcription NF-κB activity is inhibited by minocycline, a second-generation tetracycline 38, 54 Rev Nuclear export Peptides targeted against the NES domain inhibit Rev function 57 The cellular protease furin Viral assembly Peptides mimicking a conserved target sequence inhibit furin activity and thereby cleavage of the Env protein within the ER 72 HIV-1 infected cells All A Tat-Casp3 fusion protein induces apoptosis after cleavage and activation by the HIV-1 protease 79 Virus-receptor interaction and entry HIV-1 infection is initiated by binding of the virion gp120 surface subunit (SU protein) to the CD4 receptor. The SU protein is attached to the virus by a non-covalent binding to the gp41 transmembrane subunit (TM protein). Both SU and TM are proteolytically cleaved from the Envelope (Env) precursor protein by a cellular convertase, furin, within the endoplasmatic reticulum (ER). Both remain noncovalently attached and are targeted to the host plasma membrane by vesicular transport. The SU protein is responsible for receptor recognition on CD4 + T-lymphocytes and the TM protein mediates the fusion between the viral membrane and the host cell membrane [ 4 , 5 ]. Binding to CD4 induces a structural alteration in SU that exposes the binding site for a co-receptor of the chemokine family. The major co-receptors required for entry of HIV-1 are the chemokine receptor molecules CCR-5 (R5 HIV-1 isolates) and CXCR-4 (X4 HIV-1 isolates), which are used by monocytes/macrophage-tropic and T-cell tropic HIV-1 viruses, respectively [ 6 ]. When the SU protein binds to the co-receptor the result is another structural alteration exposing the N-terminal part of TM. This part, also known as the fusion-peptide, mediates the fusion between the viral and host membranes. The Env protein is also capable of mediating fusion between infected and non-infected cells by a process known as syncytium formation [ 4 , 7 , 8 ]. Current strategies are targeting particularly the CD4-SU interaction, the SU-chemokine co-receptor interaction, and the TM-mediated virus-cell membrane fusion process. The SU-chemokine co-receptor interaction CCR-5 and CXCR-4 co-receptors have specific chemokine ligands/antagonists that possess the ability to block the virus infection. The molecules that bind to the co-receptors can be divided into four categories: naturally occurring chemokines and their derivatives, peptides and small molecules (< 1 kDa), and Mabs, which recognize epitopes on for instance the extracellular domains of certain receptors. Examples of chemokine ligands (beta-chemokines) that inhibit infection of R5 isolates include RANTES, a physiological ligand for the HIV-1 co-receptors CCR3 and CCR5. RANTES is actively secreted by normal T-cells. Derivatives of this peptide have been used, including aminooxypentane (AOP)-RANTES [ 9 ], and from a recent study, N α -(n-nonanoyl)- des- Ser 1 [L-thioproline 2 , L-α-cyclohexyl-glycine 3 ] RANTES (PSC-RANTES) [ 10 ]. RANTES is an antagonist that besides having the ability to interact with CCR5 also has a downregulating effect on the co-receptor. RANTES can however induce chemotaxis and promote unwanted inflammatory side effects. Therefore AOP-RANTES was created by chemical modification of the amino terminus. This analogue does not promote any inflammatory side effects, and in addition it can prevent chemotaxis induced by e.g. RANTES. AOP-RANTES is a very strong antagonist that has a high affinity for CCR5, elicits rapid endocytosis of CCR5, and prevents recycling of the co-receptor back to the surface. PSC-RANTES is chemically identical to native RANTES except for the substitution of a nonanoyl group, thioproline, and cyclohexylglycine for the first three N-terminal amino acids of the native protein. This analogue acts in the same way, but has shown more potent in vitro antiviral activity than AOP-RANTES. Furthermore, it has successfully protected rhesus macaques from intravaginal exposure to a chimeric simian/human immunodeficiency virus containing an R5-tropic envelope of HIV-1 [ 10 ]. In addition to RANTES and its derivatives, the chemokine ligands macrophage inflammatory proteins 1alpha/beta (MIP-1alpha and MIP-1beta) also show an inhibiting effect by mediating a receptor blockade [ 8 , 11 - 13 ]. Examples of chemokine ligands that inhibit infection of X4 isolates include stromal cell-derived factor-1alpha (SDF-1alpha) and its derivatives that inhibit HIV-1 fusion and entry by minimizing the accessibility to the co-receptor on the cell surface and by inhibiting the SU-CXCR4 interaction [ 9 , 11 - 13 ]. The CCR5 amino-terminal domain is thought to play an important role in virus fusion and entry. This knowledge has been utilized in the development of anti-CCR5 Mabs whose epitopes include residues in the amino-terminal domain. Mabs of this kind strongly inhibit SU binding to CCR5 but only moderately inhibit HIV-1 fusion and entry [ 12 ]. Another type of Mab, the anti-ECL2 Mab whose epitopes include residues from one of the extracellular loops on CCR5 (ECL2), potently inhibits HIV-1 fusion and entry, but only moderately inhibits SU binding [ 12 ]. PRO 140, also an anti-CCR5 Mab, inhibits viral fusion with the cell membrane at concentrations that do not prevent the CCR5 chemokine receptor activity. It binds a complex epitope spanning multiple extracellular domains on CCR5, and although it acts as a weak antagonist it does not induce signaling or downregulation of CCR5. It is thought that the antiviral effect is exerted through a mechanism involving receptor blockade [ 14 ]. Mab 12G5 is a monoclonal antibody that recognizes an epitope on CXCR4. This epitope is also present in ECL2, and binding inhibits HIV-1 fusion [ 12 , 15 ]. A potential disadvantage of this strategy is that binding of the antibody to a receptor may trigger unwanted signal transduction [ 14 , 16 ]. Peptides, resembling the CCR5 transmembrane helices, inhibit HIV-1 replication and chemokine signaling by disrupting helix-helix interactions, which may influence the CCR5 structure [ 12 ]. T22 is a positively charged cyclic 18-mer antimicrobial peptide, which presumably inhibits SU-CXCR4 interaction by associating with the negatively charged surface of CXCR4 [ 8 , 12 ]. A truncated form of SDF-1alpha, consisting of the 16 amino-terminal residues of SDF-1alpha, also seems to possess such a blocking effect [ 12 ]. Recently, a new kind of CCR5 antagonist has been discovered in a protozoan parasite, Toxoplasma gondii [ 17 ]. This protein, cyclophilin-18 (C-18), has several potential antiviral properties including CCR5 binding, induction of the production of interleukin-12 (IL-12) from murine dendritic cells, inhibition of fusion and infectivity of R5 isolates by co-receptor antagonism and blocking of syncytia formation. Small organic molecules, such as AMD3100, potently inhibit HIV-1 replication by an interaction with residues present on one of the CXCR4 extracellular loops, ECL2, and residues within a transmembrane helix, TM4. Upon binding to these residues AMD3100 spans the main ligand-binding cavity of CXCR4, which probably constrains the co-receptor in an inactive conformation [ 12 ]. Individuals with a homozygous deletion in the gene encoding CCR5 are healthy and protected against HIV-1 transmission, which suggests that down regulation may not pose any clinical side effects. This knowledge has led to the development of strategies that directly target the mRNA encoding CCR5 or CXC4, either by ribozymes [ 6 , 18 ], anti-sense RNA [ 6 , 18 , 19 ] or RNAi [ 20 ]. The latter strategy, the siRNA approach, has led to successful blocking of HIV-1 entry, protection of cells from infection and delay of virus replication [ 21 - 24 ]. Interestingly, it is thought that single-stranded siRNAs (the anti-sense strand of a siRNA duplex) act through the same RNAi pathway, but at a later stage than double-stranded siRNA, thereby requiring less time to exert their antiviral activity [ 21 , 25 ]. The CD4-SU interaction Soluble CD4 (sCD4) is an anti-HIV-1 protein, which can be expressed and secreted from genetically engineered cells. It is a truncated form of the CD4 receptor, composed of the ectodomain that inhibits laboratory-adapted strains of HIV-1. sCD4 probably prevents the binding of the virus to the cell, by binding directly to Env, or indirectly by inducing or repressing cellular factors that influence the viral gene expression [ 18 , 19 ]. When sCD4 binds to SU it acts by extending the distance to TM, which inhibits the fusion. But when used against primary isolates, sCD4 was much less successful because of a lower affinity for sCD4. Surprisingly, some isolates became more infectious upon sCD4 treatment. An explanation for this may be that an interaction between the SU protein and sCD4 induces changes in SU, allowing it to bind the co-receptor with higher affinity or increased kinetics. In addition this interaction can eventually facilitate the fusion of HIV-1 with CD4 - cells expressing the co-receptor [ 13 ]. This has led to the development of a tetrameric version of sCD4, PRO542, in which the SU-binding region of CD4 is fused to the conserved region of human immunoglobulin IgG2. This fusion protein has a high affinity for the SU protein and has shown promising results in phase I clinical trials [ 8 , 13 ]. siRNA-directed silencing of CD4 mRNA expression has been shown to specifically inhibit HIV-1 entry and thus HIV-1 replication [ 23 , 24 ]. However, CD4 silencing in vivo may interfere with its role in normal immune function. Thus an approach targeting the CD4-binding domain of the SU protein would be more relevant. This has successfully been achieved by expressing a 0.5 kb dsRNA containing the major CD4-binding domain of the SU protein, as the target of the env gene. By this approach it has been possible to significantly suppress the expression of the HIV-1 CA-p24 antigen in human peripheral blood mononuclear cells (PBMCs) and in HeLa-CD4 + for a relatively long period of time [ 26 ]. Strategies based on the intracellular expression of antibodies specific for the HIV-1 envelope (anti-SU) have also been shown to inhibit virus replication. This strategy is based on the usage of sFvs, containing the smallest structural domain that still possesses complete antigen and binding-site specificity of the parental antibody. They are secreted into the medium where they probably act as inhibitors by direct interaction with the viral proteins [ 18 ] to neutralize the virus [ 19 ]. Cyanovirin (CV-N), an 11 kDa protein originally isolated from cyanobacteria, potently inactivates diverse strains of HIV-1. It has a high affinity for the SU protein, and when bound it inhibits the SU-CD4 interaction. CV-N possesses the advantage that even high concentrations are non-toxic and it is an extremely stable protein. CV-N has also been coupled to a cytotoxin (Pseudomonas exotoxin), thereby selectively killing HIV-1 infected SU-expressing cells [ 15 ]. The TM-mediated virus-cell membrane fusion As the SU protein binds to CD4, it initiates conformational changes in SU, making the interaction between the SU protein and the co-receptors more favorable. After attachment to the co-receptor further conformational changes occur in both the SU and TM proteins, thus weakening their interaction. During this process a transitory pre-hairpin intermediate of the TM protein is created, freeing the previously buried fusion peptide to interact with the host-cell membrane. This exposes the N-peptide and the C-peptide regions on the pre-hairpin intermediate that have been targets for several inhibiting strategies including synthetic C-peptides, N-peptides and sFvs. C-peptides are based on the C terminal end of the fusion peptide, and mimics this part of the fusion peptide when it has its correct fusogenic conformation. T-20, a 36-amino acid C-peptide, is a potent inhibitor of HIV-1 infection. It acts through a dominant negative mechanism and interacts by binding to a conserved domain on the N-peptide present in the pre-hairpin intermediate. The function of this domain is to mediate a structural change, which allows the pre-hairpin intermediate to form a fusogenic hairpin state. Binding of T-20 inhibits this process and thereby impedes fusion. Disadvantages of the C-peptide strategy are the cost of C-peptide synthesis and the relatively large amounts necessary for an antiviral effect. In addition, their size makes them non-amenable to oral routes of entry and they must be injected instead [ 13 , 27 , 28 ]. The 5-Helix is a 25 amino acid N-peptide consisting of five of the six helixes constituting the C-peptide. The peptide is presumed to inhibit fusion, through binding with high affinity to the C-peptide. However because N-peptides have a strong tendency to aggregate the inhibition could also be due to their intercalation into the TM amino-terminal coiled coil [ 27 , 28 ]. A third kind of peptides named D-peptides have also proven effective. These peptides are small 16–18 D-amino acids residues that specifically bind to three hydrophobic pockets present at the end of the N-peptide. Since such peptides are unnatural, they are resistant to proteolytic degradation, which makes them attractive for clinical use [ 13 , 27 ]. Recently, a non-neutralizing antibody directed against epitopes exposed on the fusion peptide has been reported to possess antiviral effect [ 29 ]. This antibody does not neutralize HIV-1 entry when produced as a soluble protein. However, when expressed on the cell surface as a membrane-bound sFv, it is turned into a neutralizing antibody, which markedly inhibits HIV-1 replication and cell-cell fusion by a mechanism that is thought to involve an interaction with the exposed fusion peptide. This results in inhibition of the subsequent fusion process. In the same study, this sFv was targeted into the ER and trans-Golgi network of HIV-1 susceptible cell lines where it was found to significantly block the maturation process of the viral Env protein resulting in an impairment of viral assembly. Reverse transcription and proviral integration After fusion the viral core enters the cytoplasm and the viral RNA is copied into double-stranded cDNA. This process is mediated by the viral reverse transcriptase (RT) enzyme in a complex consisting of RT, the viral genome, and a cellular tRNA 3 lys . The latter acts as primer and initiates negative strand DNA synthesis by binding to the primer binding site (PBS) region, located immediately 3' to the U5 region [ 6 , 4 ]. RT possesses three essential activities important for replication of the virus: RNA-dependent DNA polymerase (i.e. reverse transcriptase), RNase H activity (i.e. cleaves the genomic RNA in RNA/DNA hybrids during DNA synthesis), DNA-dependent DNA polymerase activity (i.e. for synthesis of the second strand of the proviral DNA) [ 6 , 4 ]. Because RT is essential for viral replication it has been one of the most popular targets. This has led to the following antiviral strategies. RT-targeted strategies Inhibiting strategies against RT involve the utilization of nucleosides and non-nucleosides. The nucleoside analogues lack the 3'-hydroxyl group, prevent the continued polymerization of the DNA chain, and are usually named nucleoside reverse transcriptase inhibitors (NRTIs). Clinically approved examples include Zidovudine (AZT), Didanosine (ddI), Zalcitabine (ddC), Lamivudine (3TC), Abacavir succinate and Stavudine (d4T) [ 8 ]. The non-nucleoside analogues, often referred to as non-nucleoside reverse transcriptase inhibitors (NNRTIs), act at the same step in the viral life cycle as the nucleoside analogous, but by a significantly different mechanism. Instead of acting as false nucleosides, the NNRTIs bind directly and non-competitively to RT in a way that inhibits the enzyme's activity. Examples of clinically approved NNRTIs include Nevirapine, Delaviridine and Efavirenz [ 8 ]. NRTIs bind to the deoxynucleoside triphosphate-binding pocket, which is formed partly by the template-primer nucleic acid and partly by the protein surfaces. NNRTIs bind to a hydrophobic pocket exclusively present in the RT enzyme of M subtype HIV-1. When used in combination they have a more pronounced antiviral effect. The RNA decoy strategy aimed at RT involves the expression of RNAs lacking the PBS region, thus preventing it from acting as template for reverse transcription. The RNA competes with HIV-1 RNA when RT makes the first jump during the first strand transfer [ 6 ]. Another decoy was designed to be co-packaged together with genomic RNA into new virions where it competes subsequently with genomic RNA for RT binding [ 6 ]. Also, a designed tRNA 3 Lys mutant containing an 11 nucleotide 3'-end complementary to the HIV-1 TAR region, shows an inhibiting effect. This mutant competes with cellular tRNA 3 Lys for the binding to RT and primes reverse transcription from the TAR region instead of the PBS region [ 6 ]. Other strategies against the RT enzyme involve the usage of small peptides, about 15–19 amino acids long, that inhibit RT activity by interfering with the dimerization process of the RT enzyme. The amino acid sequence corresponds to the so-called connection domain of RT, in particular a tryptophan-rich 19-mer sequence corresponding to residues 389–407, which efficiently inhibits viral replication [ 30 ]. Likewise, strategies based on intracellular expression of sFvs [ 7 , 18 ] and RNA aptamers [ 31 - 33 ] targeted against the RT enzyme are potent inhibitors of HIV-1 replication. The aptamers all recognize the same template-primer-binding cleft on RT. Some of these RNA aptamers have the potential to form pseudoknot-like secondary structures, which mimic the conformation of the template-primer when associated with the RT enzyme. Thus, these aptamers are termed template analogue RT inhibitors (TRTIs). Selectivity of the RNA aptamers is directly related to their three-dimensional structure. Utilization of the TRTI aptamers has the following benefits: 1) Aptamers have a unique specificity and a strong binding affinity for the RT enzyme. 2) Aptamers inhibit the RT enzyme competitively and will unlikely inhibit other viral or cellular proteins, thus minimizing the risk for any appreciable toxic side effects. 3) Since aptamers are expressed in the infected cell, the aptamers will be co-packaged into new virions and inhibit the next round of replication. 4) Because of the large interface of the aptamer-binding pocket, the risk of escape mutants is significantly reduced. Furthermore, mutations in essential binding domains, such as the template-primer-binding pocket, will likely impair the binding of the RT enzyme to the viral genome [ 31 ]. Anti-sense RNAs designed to be complementary to the Psi-gag and the U3-5'UTR-gag-env regions have been shown to inhibit RT in new virion particles. They are co-packaged together with the genomic RNA into the virus progeny, and inhibit reverse transcription by hybridizing to the genomic RNA [ 6 ]. siRNAs directed against several regions of the HIV-1 genome, including the viral long terminal repeat (LTR) and the accessory genes vif and nef have provided evidence that the viral genomic RNA, as it exists within the virion as a nucleoprotein reverse transcription complex, is amenable to siRNA-mediated degradation [ 34 ]. In addition, siRNAs targeted against the RT gene alone have shown potent inhibition of HIV-1 replication in MAGI cells [ 35 ]. Hammerhead ribozymes targeted against the HIV-1 gag region will cleave the viral RNA before reverse transcription is completed [ 6 ]. Hairpin ribozymes, designed to cleave a conserved site in the U5 region of the HIV-1 RNA can likewise inhibit replication [ 6 ]. Especially the tRNA Val -U5-ribozyme has shown promising results and is currently being tested in clinical trials. Moreover, hairpin and hammerhead ribozymes targeted against the HIV-1 pol region also show promising results [ 6 , 36 ]. In the latter strategy a hammerhead ribozyme has successfully been packaged into virions by linking it to the portion of the HIV-1 genome that provides the packaging sequence [ 36 ]. This intravirion targeting ribozyme has in the same study shown higher virus-suppressing activity than a nonpackageable counterpart. Since host tRNA 3 Lys is being packaged into new virus particles, this molecule is often used when ribozymes have to be co-packaged. An example is the tRNA 3 Lys -hammerhead ribozymes targeted against the PBS region. Besides cleaving the HIV-1 RNA, the tRNA 3 Lys -ribozyme inhibits reverse transcription by competing with host tRNA 3 Lys for RT binding and/or for the binding to the PBS sequence. Also, when bound to the PBS, the tRNA 3 Lys -ribozyme is unable to prime reverse transcription [ 6 ]. In a study closely related to the earlier mentioned CCR5 antagonist, C-18, human cyclophilin A (CyPA) has been shown to be incorporated into HIV-1 during virion assembly through interaction with an exposed proline-rich loop within the capsid domain of Gag [ 37 , 38 ]. CyPA is required for efficient viral replication but not for cell viability meaning that its cellular function is probably being compensated for by other factors. It has been proposed that CyPA enhances HIV-1 infectivity during early post-entry events, but may also be required for viral entry. The proposed molecular interaction that underlies this enhancement is the CyPA proteins ability to mask the binding site for the human host restriction factor Ref1 and thereby counteracting its inhibitory activity, allowing reverse transcription to be completed. In an attempt to reduce CyPA biosynthesis, two different anti-sense strategies were used [ 37 ]. In one approach internal CyPA exons are skipped by means of modified derivatives of U7 small nuclear RNA (snRNA). U7 snRNA is the RNA component of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone RNA 3'-end processing. By inserting appropriate anti-sense sequences into U7 snRNA it has successfully been converted from a mediator of histone RNA processing to an effector of alternative splicing. The other strategy involves the use of hairpin siRNA constructs targeting two different parts of the CyPA coding region. Both strategies greatly reduced the levels of CyPA, creating CEM-SS T-cells that sustain HIV-1 replication. The next step is the translocation of the cDNA containing capsid into the nucleus. This process is mediated by independent pathways involving either the Vpr accessory protein, the matrix protein (MA) or the integrase (IN) protein. Vpr is thought to mediate the nuclear import of the preintegration complex through the nuclear pore complex (NPC) in non-dividing cells by interacting directly with proteins in the NPC. This transfer of viral DNA is mediated by a nuclear localization signal present in the Vpr protein. Furthermore, it has been shown that Vpr is involved in arresting HIV-infected cells in the G2 phase of the cell cycle, where the virus production has been shown to reach a maximum level [ 4 - 7 ]. Integration of proviral DNA is mediated by the viral IN enzyme by a process that requires the host protein integrase interactor 1 (INI1 / hSNF5). IN consists of an N-terminal zinc finger domain, a catalytic core domain, and a C-terminal domain that is important for binding HIV-1 LTR DNA [ 39 , 40 ]. It has two enzymatic functions; DNA cleavage and insertion of the provirus into the genome of the host [ 4 , 7 ]. IN recognizes short inverted repeats (att sites) at both ends of the proviral DNA and cleaves an AT overhang at the 5' end. Then it catalyzes the non-specific cleavage of the host genome and the subsequently ligation of the 5' overhang to the cellular genome [ 4 ]. Several strategies aiming at the IN function have been reported: IN-targeted strategies IN has no known functional analogue in human cells and is therefore an appealing target for inhibiting strategies, which generally involves the usage of oligonucleotides, dinucleotides and different kinds of chemical agents, such as dicaffeoylquinic acids (DCQAs) [ 40 ] and 2,4-dioxobutanoic acid analogous [ 41 ]. The integrase binding site in the U3 LTR region of the viral DNA contains a purine motif, 5'-GGAAGGG-3'. This motif has selectively been targeted by oligonucleotide-intercalator conjugates that interact with the viral DNA through triplex formation, thus blocking the catalytic functions of the IN enzyme [ 41 ]. Disadvantages of these compounds include the low intracellular permeability and the high mutation rate of HIV-1 that may result in nucleotide substitutions in the LTR. The inhibiting effect of a dinucleotide, named pdCpIsodU, is due to its ability to interact with the catalytic core domain [ 41 ]. This molecule consists of a natural D-deoxynucleoside and an isomeric L-related deoxynucleoside joined together through a stereochemically unusual internucleotide phosphate bond, which makes the molecule resistant to 5'- and 3'-exonucleases. Through binding the molecule inhibits both the 3'-processing and the DNA strand transfer step. DCQAs are non-competitive inhibitors that act by irreversible binding to the catalytic core domain. The exact chemical mechanism for this anti-IN activity is unknown, but it is thought to be caused by a simple redox-process. Two examples are 1-Methoxy-3,5-dicaffeoylquinic acid and 3,4-Dicaffeoylquinic acid. Both are relative non-toxic [ 40 ]. Finally, 2,4-dioxobutanoic acid analogous have been reported to possess potent anti-IN activity through inhibition of the DNA strand transfer step [ 41 ]. sFvs interacting with different domains on IN have been isolated, and by fusion with a nuclease, a fusion protein is created that can interact with IN in the pre-integration complex, leading to cleavage of proviral DNA. Likewise IN-specific sFvs have been shown to be inhibitory to HIV-1 replication [ 7 , 18 ]. Finally, siRNAs targeted against the capsid protein, p24-siRNA, is thought to interact with the gag gene in the unspliced viral RNA when present in the cytoplasm. Thereby, the viral RNA genome is cleaved before integration occurs [ 23 , 24 , 42 ]. HIV-1 transcription Transcriptional regulation of HIV-1 gene expression is controlled by co-operative and cell-specific interactions between several host cells transcription factors, including AP-1, NF-κB, NF-AT, NF-IL-6, CREB, IRF, Sp1, LEF-1/TCF-1α, Ets-1 and USF, and the viral Tat protein [ 5 , 7 , 43 ]. The Tat protein recognizes a stem-loop structure, the trans-activation responsive element region (TAR), located in the 5'-end of the primary transcript (R region). Tat recruits a cellular co-factor, positive transcription elongation factor b (P-TEFb), composed of human cyclin T1 (hCycT1) and CDK9 (a CTD kinase). The hCycT1 component binds to the activation domain of Tat thereby increasing the affinity for TAR. This results in the formation of a Tat/TAR complex. Next, CDK9 phosphorylates the carboxy-terminal domain of the host cell RNA polymerase II, which stimulates the elongation process and thereby the overall transcriptional efficiency [ 4 , 44 ]. The Tat/TAR interaction is essential for activation of HIV-1 transcription and is therefore a popular target for inhibiting strategies. Another reason for choosing strategies directed against this step is that the Tat-TAR interaction is highly conserved. Thus the chance for development of escape mutants is very low, due to the fact that mutations in either Tat or TAR will cause an impaired interaction between them and thereby abolish HIV-1 replication. One strategy is to express a Tat protein that displays a transdominant negative phenotype, which can inhibit the replication of HIV-1. These proteins act as competitors for Tat binding to an essential substrate or co-factor, or alternatively by associating with wild-type monomers to form an inactive mixed multimer. Examples include Tat proteins containing mutations in the activating domain, the protein-binding domain, or in the TAR binding domain [ 7 , 18 , 45 ]. An obvious disadvantage of this strategy is, as mentioned earlier, the mutants' ability to recruit co-factors important for maintaining of a normal cellular function. Tat function can also be impaired by using a single-chain antibody, sFv-Tat. When sFv-Tat interacts with the Tat protein, it restrains Tat in the cytoplasm, thus hindering its transcription-regulating function in the nucleus [ 7 , 18 ]. Several studies have shown promising results in blocking the interaction of cellular TAR RNA-binding proteins and viral Tat protein to TAR RNA. For instance these include a study in which a compound termed TR87 directly competes with Tat for binding to TAR [ 46 ], and a study involving pyrrolo [2,1-c][1,4] benzodiazepine-oligopyrrolo hybrids, which appear to interrupt protein/TAR RNA interactions and Tat-induced LTR-driven HIV-1 transcription [ 47 ], and finally a study were two aromatic polyamidines carrying a halogen atom, termed TAPB-Br and TAPP-Br, have demonstrated the potential to inhibit cellular and viral protein-TAR RNA interactions [ 48 ]. An inhibiting effect has also been observed using anti-sense-, nuclease-, or siRNA-based strategies directed against tat mRNA [ 6 , 7 , 35 , 45 , 49 ]. Notably, a recent study has demonstrated that tat siRNA delivered as pre-miRNA precursor is 80 % more effective than tat siRNA expressed as conventional short hairpin RNAs (shRNAs) [ 50 ]. Finally, anti-sense RNAs and nucleases targeting the TAR element have also shown promise as antivirals [ 5 - 7 , 45 ]. A Tat-nuclease fusion protein has been engineered by fusing the HIV-1 TAR RNA binding domain of HIV-1 Tat with the RNase H domain of HIV-1 RT. Since TAR is present at the 5' and 3' ends of all HIV RNAs, this Tat-nuclease can recognize and cleave all HIV-1 RNA transcripts. The Tat protein cycles in and out of the nucleus and the cleavage of HIV-1 transcripts should therefore take place both in the nucleus and in the cytoplasm [ 5 ]. TAR decoys represent another example of a suitable strategy. These sense RNAs act by interacting with Tat-containing RNA polymerase II transcription complexes that assemble on the HIV-1 promoter [ 7 ]. In addition the TAR decoy RNAs solely recruit the Tat protein and the cellular co-factor, P-TEFb, which is necessary for Tat-mediated transactivation [ 18 ]. To make this strategy more effective, the development of polymeric TAR decoys has been accomplished. Constructs with up to 50 TAR elements have been reported, but unfortunately these constructs also recruit essential functional cellular co-factors [ 6 , 18 ]. A TAR decoy based on the element of HIV-2 (TAR-2) has been shown to suppress HIV-1 replication more effective that the decoy based on HIV-1. The explanation for this is that the TAR-2 structure possesses three separate loop regions and may therefore more effectively compete with the single stem-loop structured TAR in HIV-1 for loop-binding cellular factors [ 51 ]. Besides targeting the Tat encoding regions, siRNAs have been directed against the Gag [ 23 , 24 , 42 ] and Nef [ 23 , 34 ] encoding regions. Both siRNAs show antiviral effects. The gag-targeted siRNA (p24-siRNA) is identical to the p24-siRNA utilized when inhibiting the pre-integration step and acts in the same manner. Since the nef gene is located in the 3' end of the HIV-1 genome and in many of the viral transcripts, a siRNA directed against the Nef encoding region will reduce the number of viral transcripts. Also a 3'-LTR directed siRNA has shown potently to suppress viral replication [ 42 ]. The 3'-LTR region was chosen, as it is in the noncoding sequence before the poly(A) tail, except for the Nef encoding RNA. Thus, by this specific siRNA approach it is possible to target both spliced and unspliced RNA produced from the provirus, whereas the p24-siRNA approach only targets the unspliced viral RNA. By combining these siRNAs a synergistic effect has been observed [ 23 , 24 , 34 , 42 ]. The high specificity of the RNAi approach also makes it vulnerable to inactivating mutations in the viral genome as was observed in a recent study [ 52 ]. Another promising strategy involving siRNAs includes targeting of the NF-κB p65 subunit [ 35 ]. The NF-κB p65 subunit is a key component for NF-κB transcriptional activation of HIV-1. During the early phases of HIV-1 infection in activated T lymphocytes, NF-κB binding to the HIV-1 LTR serves to stimulate the generation of at least some full-length transcripts for synthesis of Tat, which then stimulates the transcriptional elongation process. This is supported by the observation that siRNAs targeted against the NF-κB p65 subunit show a decrease of HIV-1 replication in MAGI and Jurkat cells. However, the NF-κB proteins are also critical for the regulation of immune function. They regulate the expression of a variety of genes encoding cytokines and cytokine receptors, chemokines, cell adhesion molecules, and cell surface receptors that are critical for T- and B-cell function. Therefore further studies are required before p65-siRNAs can be used in clinical trials [ 3 , 35 , 49 ]. Finally, a siRNA mediated knockdown of cellular P-TEFb has surprisingly shown to decrease HIV-1 transcription and viral replication without being lethal to the cell. It seems that there is a critical threshold of P-TEFb kinase activity that is required for cell viability and Tat transactivation. Moreover, it is suggested built-in intracellular mechanisms allow cells to cope with changes in P-TEFb protein levels [ 53 ]. Recently it has been reported that minocycline (MC), a second-generation non-toxic tetracycline, possesses the ability to inhibit NF-κB transcriptional activation and thereby viral replication in microglia [ 54 ]. These resident brain macrophages play a central role in AIDS dementia, as they are the primary targets of productive infection in the brain. Besides interrupting LTR activation, this agent also seems to influence the production of cytokines, chemokines, and other substances implicated in AIDS dementia. It appears that MC may act by increasing NF-κB complex formation, resulting in inactive homodimers. This suggests that MC possess the ability to suppress both viral production and inflammation. HIV-1 mRNA splicing and nuclear export Viral gene expression can be divided into an early and late phase, which is Rev-independent and Rev-dependent, respectively. In the early phase the newly transcribed mRNA is spliced by the cellular splicing machinery into multiply spliced transcripts, which mainly produces the Tat, Rev and Nef proteins. When Rev has accumulated to a critical level the mRNA production shifts from multiply spliced to the singly spliced and unspliced transcripts, characteristic of the late phase of gene expression. Rev contains an RNA binding motif that directly interacts with stem-loop IIB located within an RNA multi-stem-loop secondary structure, termed the rev response element (RRE), which is present in the env gene of all incompletely spliced viral mRNAs. The RRE can accommodate the binding of at least 8 Rev molecules, and at a certain threshold concentration of Rev protein in the nucleus, functional Rev/RRE complexes are formed, which greatly stimulate the export of unspliced and singly spliced RNA to the cytoplasm where translation can proceed. Nuclear export is mediated by cellular co-factors termed exportin 1 and Ran-GTP, which interacts cooperatively with the Rev nuclear export signal (NES) sequence [ 55 ]. The nuclear import of Rev is mediated by the cellular co-factor importin-beta that interacts directly with a NLS sequence in Rev. The host proteins B23 and p32 also interacts with the NLS region and may be involved in nucleolar localization [ 4 , 5 , 56 - 58 ]. The fundamental and essential function of Rev has made it a popular target for therapeutic development [ 57 ]. Fig. 2 illustrates the structure of proviral DNA and the different RNA species. Figure 2 Schematic representation of the HIV-1 provirus and the different RNA species. Gag; group specific antigen, Gag-Pol; group specific antigen-polymerase, Env; envelope, Tat; trans-activator of transcription, Rev; regulator of expression of virion proteins, Nef; negative effector, Vif; virion infectivity factor, Vpr; viral protein r, Vpu; viral protein u, LTR; long terminal repeat. Many of the used strategies are based on inhibiting the Rev/RRE interaction. Examples include Rev TNPs, RRE RNA decoys, anti-sense Rev/RRE RNAs, siRNAs, sFvs, nucleases, ribozymes, aptamers, chimeric proteins, and small inhibiting molecules. The most well characterized Rev TNP is the RevM10 protein, which contains amino acid substitutions within the NES region. The ability of RevM10 to form multimeric structures and to interact with RRE is not hindered by these mutations, but the interaction with cellular co-factors is prevented leading to inhibition of Rev function [ 7 , 18 , 25 , 57 - 59 ]. Making deletions in the NLS sequence generates another kind of Rev TNP, the Rev38. The result is a mutant that accumulates in the cytoplasm where it sequesters the wildtype Rev protein by formation of inactive oligomers, thereby hindering the transport of Rev into the nucleus [ 18 , 57 ]. Likewise, Rev mutants lacking the ability to form multimeric structures (RevSLT26 and RevSLT40) are effective inhibitors. Of the mentioned TNPs, RevM10 is the most potent inhibitor that has been used in clinical trials [ 57 ]. RRE RNA decoys act by recruiting the Rev molecules and thereby hindering their interaction with the viral transcript. Potent inhibition has been achieved by overexpression of a 45 nucleotide chimeric tRNA-RRE transcript [ 7 ]. This type of decoy unfortunately also binds essential cellular co-factors, which has led to the design of minimal RNA decoys that only contains the Rev binding site. An example is a 41 nucleotide RRE SLIIAB RNA decoy that has been used in clinical trials [ 6 ]. sFvs targeted against Rev potently recruit Rev proteins in the cytoplasm, which accelerates the degradation of Rev [ 7 , 18 , 25 , 57 ]. In a comparative study the effect of monoclonal sFv targeting either the NES region or the C-terminal region of Rev was compared. The NES-specific sFv demonstrated the best antiviral effect, even though the binding affinity of the C-terminal sFv for Rev was significantly higher [ 57 ]. Additional strategies for inhibition of Rev function include, 1) RNA aptamers, possessing a higher affinity for Rev than the RRE sequence, and which recognize other epitopes than the natural RNA binding site on Rev [ 57 ], 2) peptides that recognize the NES domain [ 57 ], 3) a dominant negative mutant form of Sam68 (Src-associated protein in mitosis), whose natural function is to interact with RRE and thereby partially substitute or synergize with Rev in RRE-mediated gene expression. In particular, a C-terminally deleted mutant of Sam68 inhibits not only Sam68 transactivation of RRE, but also Rev function [ 60 ]. Unlike RevM10, which competes with wildtype Rev for binding to RRE in the nucleus, this Sam68 mutant is mainly cytoplasmic, and binds RRE very poorly. However, it retains the ability to bind Rev and the mechanism may involve trapping of Rev in the cytoplasm by direct protein-protein interaction. It is thought that the complex formation of Sam68 and Rev has a masking effect that inactivates the Rev NLS domain. Since the Sam68 NLS domain is deleted in the mutant it will not be able to substitute for the missing Rev NLS domain, thus the HIV-1 replication is inhibited. 4) chimeric proteins. One example is a construct where the Rev protein is covalently attached to a mutant form of the NS1 protein from the Influenza A virus (NS1RM-Rev). It is thought that the fusion protein and wildtype Rev form mixed oligomers, and due to the nuclear retention activity of NS1, which is dominant over the Rev-mediated nuclear export, it results in inhibition of nuclear export of viral transcripts. NS1RM-Rev has an antiviral effect equal to that of the RevM10 mutant [ 7 , 57 ]. As with inhibition of the Tat/TAR interaction, the anti-sense and ribozyme strategies can also be targeted towards the Rev/RRE interaction. Anti-sense directed against RRE will inhibit Rev binding, whereas anti-sense directed against the Rev encoding region will hinder the expression of Rev protein [ 6 ]. The anti-sense RNAs can be either unmodified or modified RNAs. E.g. a synthetic phosphorothioate oligodeoxynucleotide targeting Rev mRNA has antiviral activity in chronically infected cells [ 25 ]. Unfortunately, this type of anti-sense RNA strategy has shown limited success as a therapeutic agent because of unsolved problems such as efficacy, cell permeability, delivery and cost. Ribozymes targeting RRE or the Rev encoding region hinder viral replication by cleaving the targeted RNA [ 6 , 7 ]. By fusing the Rev protein with a nuclease it is possible to create a nuclease with affinity for the RRE and which therefore has the potential ability to specifically cleave all HIV-1 RNAs carrying the RRE sequence [ 5 ]. Also, siRNAs directed against the rev transcript potently inhibit virus replication [ 49 , 61 ]. Finally, strategies based on small molecules that either interact with host cellular proteins or bind to RRE, have also been applied to inhibit Rev function. Due to the ability to interact with cellular proteins, these molecules are often cytotoxic and therefore not usable for eventual clinical trials. Nevertheless they are included here because less toxic derivatives could be developed in the future. An example is Leptomycin (LMB), an antibiotic agent that interacts with the cellular protein CRM1, blocking the binding to the Rev NES domain. A clear disadvantage of LMB is, besides long-term toxicity, its ability to block the transport of other proteins with a NES domain [ 25 , 56 , 57 ]. An overexpressed truncated version of the nucleopurine, Nup214/CAN, delta-CAN, has shown a closely related mechanism of inhibition. Delta-CAN retains the skill to interact with CRM1 and inhibits Rev function in the same way [ 56 ]. Aminoglycoside antibiotics, such as neomycin B and derivatives have shown antiviral effect by binding to RRE and thereby hindering the Rev/RRE interaction. The binding of aminoglycoside antibiotics to RNA is very unspecific, and together with a low selectivity, this drug is unfortunately highly toxic for humans [ 25 , 57 ]. The intercalating dye, pyronin Y, completely blocks the formation of the Rev/RRE complex in vitro. But since pyronin Y also intercalates in DNA, the result is an elevated level of cell death. Other intercalating agents, derivatives of diphenylfuran, have been reported to inhibit the Rev/RRE interaction by causing a conformational change in RRE. These compounds possess the same disadvantages as pyronin Y [ 25 ]. HIV-1 translation As illustrated in fig. 2 translation of the non-spliced RNA and single-spliced RNA result in the Gag and Gag-Pol proteins, and in the Vpu, Vif, Vpr and Env proteins, respectively. Translation of the completely spliced RNA results in the Tat, Rev and Nef proteins [ 4 , 6 - 8 ]. Designed anti-sense RNAs directed against sequences located in the 5' UTR of all HIV-1 mRNAs, would be able to hinder the ribosome-complex in completing the translation process and thereby inhibit the protein synthesis [ 6 ]. Hairpin ribozymes directed against the U5 region have shown similar antiviral responses [ 6 , 7 ]. A study involving nef shRNAs corresponding to nef-derived miRNAs, which recently have been demonstrated to be produced in HIV-1-infected cells [ 62 ], shows the potential to efficiently block RNA stability or mRNA translation. This may indicate that HIV-1 possess the ability to regulate its own replication. Interestingly, another study has shown that HIV-1 putatively encodes five candidate pre-miRNAs, which potentially could target a large number of cellular transcripts indicating that HIV-1 moreover may have the potential to regulate the cellular milieu [ 63 ]. Viral assembly, release and maturation The virus particle is assembled at the plasma membrane. In this process the Gag and Gag-Pol polyproteins interact with each other by protein-protein interaction, most probably via the capsid (CA) protein domain [ 64 ]. The viral genome is packaged in a process in which the packaging signal, Psi, is recognized by the nucleocapsid (NC) protein domain of the Gag protein [ 65 ]. Another important function of the NC domain is to mediate the formation of the RNA dimer via a palindromic sequence in the dimer linkage structure (DLS) sequence, which is located in the Psi sequence [ 4 , 6 ]. In addition several cellular tRNAs are packaged. The assembly of the virus particle is partly regulated by the Vpu and Vif proteins. The primary function of the Vpu protein is to mediate the release of the virus particle from the cell surface, by selectively targeting the CD4 protein to a degradation pathway in the endoplasmic reticulum (ER). This permits the release of Env protein from the ER, which may otherwise be complexed with the CD4 protein, and further processing of the Env protein can then proceed [ 4 , 5 ]. A current thought is that Vif, besides influencing the late stages of virion assembly, may also block premature processing of Gag precursor protein by the HIV protease (Pro). This kind of temporal control of Gag processing ensures the availability of the CA, MA and NC peptides, when the assembly of the viral components takes place at the plasma membrane [ 4 , 66 , 67 ]. In addition, several studies have recently suggested that Vif may possess another important function in which it acts by overcoming the antiviral activity of a cellular cytidine deaminase, APOBEC3G (CEM15) that induces hypermutations in newly synthesized HIV-1 DNA [ 68 , 69 ]. The mechanism by which Vif inhibits APOBEC3G function is unclear, but it is thought to form a complex with APOBEC3G, thus preventing its viral encapsidation. Furthermore, Vif can target APOBEC3G for degradation via the ubiquitin-proteasome pathway. After virus budding from the cell surface, maturation of the virus particle proceeds. At this stage in the virion life cycle the Gag and Gag-Pol polyprotein are proteolytically cleaved by the protease domain of Gag-Pol precursors. Cleavage of Gag results in the MA, CA, NC and CEL viral proteins. The MA proteins forms a matrix under the viral envelope, the CA proteins condenses to form a conical core surrounding the NC-coated RNA genome and the CEL protein is thought to associate with the Env protein and, in addition to mediating packaging of Vpr. Cleavage of the Gag-Pol polyproteins, which is made by ribosomal frameshifting during translation of unspliced mRNA, results in the enzymatic proteins IN, RT and Pro. Both the Gag and Gag-Pol precursors associate with the Env protein by protein-protein interactions between the MA domain and the TM protein. After maturation the virus is ready for another round of infection [ 4 ]. Strategies directed against this final step in the viral life cycle include RNA decoys, TNPs, chimeric proteins, anti-infectious cellular proteins, sFvs, nucleases, anti-sense RNAs, ribozymes and peptides. Viral assembly The packaging signal Psi is a highly conserved RNA sequence and is therefore an obvious target for inhibition strategies. The Psi region contains four stem-loops located near the 5' major splice donor and the start of the gag open reading frame and is essential and sufficient for RNA packaging [ 45 ]. RNA containing a 1.43 kb region anti-sense to the Psi-gag region has been reported to inhibit packaging of genomic RNA and thus HIV-1 replication with a higher efficiency than the RevM10 mutant [ 6 , 7 , 45 ]. An antiviral effect has also been observed by targeting anti-sense RNAs against the Psi sequence, which act by hindering the NC domain of the Gag protein in recognizing the packaging signal. Likewise, ribozymes directed against the packaging signal have also been shown to have a significant effect on viral replication [ 7 , 6 ]. Since the Psi element is recognized by the NC domain, one strategy is to design a NC-nuclease fusion protein, which will recognize and cleave all unspliced HIV-1 RNAs. The cleavage process occurs in the cytoplasm and this will also inhibit the production of the Gag and Gag-Pol fusion proteins [ 5 ]. Expression of the gag-gene has also been reduced by using anti-sense RNAs complementary to the 5'-leader-gag region or by using ribozymes directed against the gag transcripts [ 7 , 18 ]. The ability of the NC domains to recognize the genomic RNA can be blocked with RNA decoys containing the Psi sequence. These decoys may form dimers with HIV-1 RNA and thereby compete with HIV-1 RNA dimers for packaging into new virus particles [ 6 , 45 ]. The structural proteins, Gag and Env, form multimeric complexes during viral assembly. By means of different kinds of Gag TNPs it has been possible to inhibit this step. Besides inhibiting viral assembly, these Gag TNPs also interfere with the viral release process, the uncoating of the viral genome and the reverse transcription [ 18 ]. A limitation of using Gag TNPs is that the gag-gene contains an inhibiting sequence, the CRS, which hinders the expression of the gag-gene if Rev is missing [ 4 , 6 , 18 ]. The potential effect of Env TNPs has also been tested, but this strategy shows relatively low antiviral activity when compared to Gag TNPs [ 18 ]. Given that one function of the Vif protein is to block early processing of the Gag protein, it has inspired the development of a Vif TNP that is missing the blocking function. Applying this protein means that the virus assembly is impaired, but a clear disadvantage is that HIV-1 has the potential to evolve escape mutants [ 66 ]. Similar inhibition has been observed by directing anti-sense RNAs against the Vif encoding region [ 70 ]. Another strategy relies on the INI1 protein that is the only known host protein directly interacting with HIV-1 integrase. A minimal integrase-binding fragment of INI1, S6, comprising amino acids 183–294, potently inhibits HIV-1 assembly, particle production and replication in a transdominant manner. This inhibiting effect results from direct interaction of the S6 protein with the integrase domain within the Gag-Pol polyprotein. When the S6 protein binds to integrase it is thought to interfere with proper multimerization of Gag and Gag-Pol by steric hindrance. In addition it affects maturation, blocks an interaction of the cellular assembly machinery with Gag-Pol and mediates the mislocalization of viral proteins into different sub-cytoplasmic compartments of the cell. Besides being non-toxic, another favorable feature of S6 is that it is unlikely to be immunogenic, because it is a truncated form of a host protein. In addition, virions with mutations in the S6 binding site will also be defective for interaction with INI1. This minimizes the risk for the development of HIV-1 escape mutants [ 39 ]. Recently, a specific inhibition of virus budding was demonstrated by overexpression of an amino-terminal fragment of tumour susceptibility gene 101 (Tsg101) [ 71 ]. The role of Tsg101 is to participate in the endocytic trafficking pathway. It is presumed to bind to the Gag polyprotein and subsequently mediate the transport into multivesicular bodies (MVBs), which then carry their cargo towards the cell surface. By targeting an intracellular sFv specifically against the CD4 binding region of the SU protein, it has been possible to make cells temporarily resistant to HIV-1 infection. This sFv, named sFv105, acts by binding to the Env protein, and traps Env in the ER. This prevents the maturation process in which Env is cleaved into the SU-TM proteins. As a result, Env is prevented from reaching the cell surface [ 7 , 18 ]. Another way to inhibit the maturation of Env is by inhibiting the cellular protease furin [ 72 ]. Furin is a member of the mammalian subtilisin-related proprotein convertases that mediate cleavage of the Env protein at a conserved Arg-Glu-Lys-Arg sequence. Peptides that mimic this sequence have been reported to block furin activity. Especially, a polyarginine peptide has shown promising results without showing any toxic side effects on cultured cells ex vivo or in mice in vivo . Furthermore, anti-sense RNA approaches directed against the Env message also belong to the applied strategies [ 70 ]. Multitarget ribozymes that target different sites in the SU sequence also exert antiviral effects. Some of these designed ribozymes bind and cleave up to nine different conserved regions in the SU sequence [ 7 ]. A Gag-nuclease fusion protein can be packed into new virions and thereafter, in the proceeding rounds of infection, efficiently cleave the viral genomic RNA. Since the Gag protein has many essential functions it is unlikely that HIV-1 will develop escape mutants when using the Gag-nuclease. Vpr- and Nef-nuclease fusion proteins also seem to cleave viral RNAs, either during or after viral assembly [ 5 , 7 ]. Viral release A Nef TNP has shown an antiviral effect by inhibiting down-regulation of e.g. the CD4 cell surface protein. It is thought that CD4 may interact with the Env protein present on new viral particles, thus hampering viral release from the cell surface [ 66 ]. Likewise, anti-sense RNAs and ribozymes directed against a conserved 14 nucleotide region in the nef-gene also possess an antiviral effect [ 7 ]. By overexpressing different kinds of CD4 variants in the infected cell, it is possible to inhibit virus budding. The reason is most likely that the CD4 variants possess the ability to trap and restrain new virions in the cell [ 19 ]. Viral maturation The HIV-1 protease plays an important role in virus maturation. As mentioned earlier, the HIV-1 protease cleaves the Gag and Gag-Pol polyproteins to form the structural and enzymatic proteins. Consequently, the protease is a potent target for inhibiting strategies. The current strategies involve protease inhibitors that bind to the active site of the HIV-1 protease and thereby inhibit processing of the Gag and Gag-Pol polyprotein precursors. This results in immature and noninfectious viral particles. The HIV-1 protease is an aspartyl protease and inhibitors have been designed that optimally bind to the catalytic aspartate residues and additionally to the water molecule that is critical for enzymatic action. The inhibitors are transition state analogues that bind the enzyme much more tightly than the natural substrate, making them competitive enzyme inhibitors. Examples of approved protease inhibitors include Saquinavir, Indinavir, Ritonavir, Nelfinavir and Amprenavir [ 8 , 11 , 73 ]. Other strategies that target Pro involve the application of Pro TNP [ 7 ], and the overexpression of chimeric Vpr protein in which the C-terminal region is fused to several cleavage sites recognized by the protease. This will overwhelm the protease activity by a competitive mechanism and impair protease function [ 7 , 74 ]. Finally, anti-sense RNAs targeting the Pol coding region inhibit this last step in the viral life cycle [ 70 ]. Combination of antiviral strategies By combining the different antiviral strategies, the effectiveness can be increased and the chances of generating escape mutants will be minimized. Examples of combination therapies include ribozymes combined with decoy or anti-sense RNAs [ 6 , 75 ], and decoy RNAs combined with anti-sense RNAs [ 6 , 76 ]. An autoregulated dual-function anti-Tat gene is an example of the latter strategy, in which both a polymeric TAR and an anti-sense Tat are combined in one expression unit [ 76 ]. To accomplish gene expression in HIV-1 infected cells, a double-copy retroviral vector, in which gene expression is driven by the HIV-1 LTR, is used. By this approach anti-Tat gene expression is upregulated only in HIV-1 infected cells. The RT and Pro inhibitors are preferentially not used alone to avoid the risk of generating viral escape mutants. By combining the different kinds of inhibitors (usually a combination of two nucleoside analogues with either a protease inhibitor or a non-nucleoside analogue) significant inhibition of HIV-1 is achieved. This combination strategy is also known as HAART (highly active antiretroviral therapy). Because nucleoside and non-nucleoside analogues act on two different positions on the RT enzyme they will not compete for binding and when used in combination they exhibit a more potent effect. The disadvantage concerning this strategy is the relative strong toxic effects related to these RT drugs. Another problem arises if the prescribed treatment is not exactly followed and resistant viral mutants emerge [ 31 ]. When combining strategies involving ribozymes and RNA decoys one can obtain better results than by using one of the strategies. This is clearly illustrated by the tRNA Val -RRE-SLII-U5 hairpin ribozyme, in which SLII (stem loop II) contains the Rev protein-binding site that acts as a decoy [ 6 ]. In an attempt to interfere with two essential HIV-1 activities at the same time, a double transdominant negative Tat-Rev fusion protein, Trev, has been designed. This fusion protein inhibits both Tat transactivation and Rev mediated nuclear transport [ 18 ]. A similar designed fusion protein, Tev, contains the RNA binding domains of both Tat and Rev, and can thus target both TAR and RRE within the HIV-1 RNA. Furthermore, a nuclease was fused to the Tev protein. The result is an inhibition of both early and late viral gene products. Tev contains a NLS and is therefore predicted to act primarily within the nucleus [ 5 ]. The combination of an anti-Rev sFv, which targets the Rev activation domain, and a ribozyme that targets RRE, or an RRE RNA decoy, which recruits the Rev molecules, has also shown good results [ 7 ]. Promising results have also been described by using a vector expressing three products, the U5 ribozyme, a ribozyme targeted against the Env/Rev encoding regions, and a RRE decoy respectively [ 18 ]. Strategies based upon suicide genes Conditional expression of suicide genes in cells infected with HIV-1, e.g. by expression from a Tat dependent promoter or under Rev dependent control, has been designed in different versions. Examples of suicide gene approaches include engineering cells with a diphtheria toxin A-chain (DT-A) gene, a cytosine deaminase gene, a herpes simplex virus (HSV) thymidine kinase (tk) gene, and a herpes simplex shutoff (vhs) gene. DT-A is a very effective cellular toxin that kills cells by blocking the protein synthesis via the ADP-ribosylating elongation factor 2 [ 77 ]. Cytosine deaminase mediates cell death through the conversion of 5-fluorocytosine to the potent cytotoxic agent 5-fluorouracil [ 18 ], and the HSV thymidine kinase mediates cell death by metabolizing nucleoside analogues, such as Ganciclovir and Acyclovir, into toxic analogues [ 7 , 18 ]. The latter strategy has been further explored in a study involving a live-attenuated form of HIV-1 in which the nef gene has been deleted and instead engineered to express the thymidine kinase gene [ 78 ]. This marked live-attenuated virus vector may be useful to accrue baseline information on the immunological benefits of a replicating vaccine. The safety profile of such a vaccine vector is supported by the possibility to remove cells harboring integrated proviral genomes if necessary. Another approach involves the design of a hybrid molecule consisting of the human CD4 and a modified version of the Pseudomonas exotoxin A (CD4-PE40). This molecule binds to infected cells by a CD4-SU interaction at the cell surface and, after uptake, the exotoxin inhibits protein synthesis, thus leading to cell death [ 7 ]. Finally, the last example involves a modified apoptosis-promoting caspase-3 protein, Tat-Casp3, which acts by cleavage of the inhibitor of caspase-activated DNase, resulting in the activation of caspase-activated DNase and, ultimately, cell death. In this design the endogenous cleavage sites have been substituted with the HIV proteolytic cleavage sites, and the Pro domain of the modified Casp3 protein was removed and substituted with the Tat transduction domain. Hence, the fusion protein is only activated by the HIV protease in infected cells, resulting in apoptosis, whereas in uninfected cells it remains in the inactive zymogen form. Tat-Casp3 proteins may also be packaged inside the virion and kill the virion after it buds from the cell and/or initiate apoptosis immediately after subsequent infection of a cell [ 79 ]. Conclusion In spite of the astonishing diversity of methods developed as antivirals against HIV-1, still many problems remain. Perhaps the most difficult problem to solve is the remarkable ability of HIV-1 to evade the different inhibiting strategies. The selection pressure enforced by the treatment may result in the selection of escape mutants that are more pathogenic than the original virus. For instance: Blocking the interaction with CCR5, which is the primarily used co-receptor, could result in usage of both the CCR5 and the CXCR4 co-receptors or CXCR4 alone. The outcome will be an accelerated reduction of CD4 + T-cells and thereby a progression of the disease [ 80 ]. The risk of evolution of a virus variant that uses new co-receptors is not unthinkable. To obtain long-term inhibition and to avoid escape mutants, it is necessary to combine the different strategies, so that several steps in the viral life cycle are inhibited at the same time. RNAi is a very promising novel approach that in principle will provide a large number of new targets that may be combined, but unfortunately one of the biggest hurdles is the in vivo delivery problem, which needs to be addressed. A gene therapy approach may be used to make hematopoietic stem cells resistant to HIV-1, which could eventually lead to (partial) restoration of the immune system. In spite of the advanced technology used in the different virus intervention strategies and the rapidly growing knowledge about the molecular biology of HIV-1, it has not yet been accomplished to block HIV-1 replication completely. Hopefully, scientists will succeed to push the balance of the virus-host battle in the right direction so that the immune system will be able to handle the remaining viruses, or vaccine strategies may be added to clear the virus. Independently of the path taken, it will most likely require that the latent reservoirs of virus are activated to make them more vulnerable to treatment. A combination of these directions may eventually lead to a complete eradication of the virus in infected patients. List of abbreviations used Aids; acquired immunodeficiency syndrome HIV-1; human immunodeficiency virus type 1 shRNA; short hairpin RNA siRNA; small interfering RNA RNAi; RNA interference miRNA; microRNA dsRNA; double stranded RNA Gag; group specific antigen CA; capsid protein MA; matrix protein NC; nucleocapsid protein CEL; core-envelope-link protein Gag-Pol; group specific antigen-polymerase RT; reverse transcriptase IN; integrase Pro; protease Env; envelope SU; surface protein TM; transmembrane protein Tat; trans-activator of transcription Rev; regulator of expression of virion proteins Nef; negative effector Vif; virion infectivity factor Vpr; viral protein r Vpu; viral protein u TAR; trans-activation responsive element RRE; rev response element Psi; packaging signal DLS; dimer linkage structure PBS; primer binding site LTR; long terminal repeat R; repeat U5; unique 5' U3; unique 3' ER; endoplasmatic reticulum NES; nuclear export signal NLS; nuclear localization signal TNP; transdominant negative protein sFv; intracellular single-chain antibody Mab; monoclonal antibody TRTI; template analogue RT inhibitor NRTI; nucleoside reverse transcriptase inhibitor NNRTI; non-nucleoside reverse transcriptase inhibitors HAART; highly active antiretroviral therapy SELEX; systematic evolution of ligands by exponential enrichment DCQA; dicaffeoylquinic acid LMB; leptomycin Tsg101; tumour susceptibility gene 101 MVB; multivesicular body DT-A; diphtheria toxin A-chain tk; thymidine kinase RANTES; regulated upon activation, normal T-cell expressed and secreted beta-chemokine PBMC; human peripheral blood mononuclear cell CV-N; Cyanovirin CyPA; human cyclophilin A Competing interests The author(s) declare that they have no competing interests. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553987.xml |
544551 | The you Gene Encodes an EGF-CUB Protein Essential for Hedgehog Signaling in Zebrafish | Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you- class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you -class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates. | Introduction The coordination of growth, proliferation, and differentiation during development requires transmission of information in the form of extracellular signals. Hedgehog signaling is of fundamental importance in the development of a wide variety of tissues and organ systems. Much of the initial functional analysis of Hedgehog signaling focused on the patterning of Drosophila larval segments and imaginal discs, dorsoventral patterning of the vertebrate neural tube, and anterior–posterior patterning of vertebrate limbs; in addition, many recent studies have illuminated the widespread and conserved role of Hedgehog signaling in development (reviewed in [ 1 ]). Misregulation of Hedgehog signaling has been implicated in several diseases and developmental abnormalities, including basal cell carcinoma [ 2 , 3 , 4 ], medulloblastoma [ 5 , 6 , 7 ], pancreatic cancer [ 8 ], and holoprosencephaly [ 9 , 10 ]. After release from signaling cells, the activity and distribution of Hedgehog proteins are modulated by a variety of factors in the extracellular environment. In Drosophila , diffusion of lipid-modified Hedgehog proteins is dependent on the action of tout velu, a gene involved in the synthesis of heparan sulfate proteoglycans [ 11 , 12 ]. The diffusion of Hedgehog is also attenuated via sequestration by its receptor, Patched [ 13 ]. In vertebrates, Hedgehog proteins may be further regulated by binding to the growth-arrest specific gene product Gas1 [ 14 ], and Hedgehog-interacting protein Hip1, which is itself induced by Hedgehog signaling [ 15 ]. Moreover, the ability of Hedgehog proteins to diffuse over significant distances in the developing vertebrate limb bud appears to depend on the cholesterol modification of the Hedgehog protein; this modification may facilitate the assembly of Hedgehog proteins into a multimeric structure, perhaps conferring increased stability or mobility [ 16 , 17 ]. Genetic and biochemical evidence suggests that the low-density receptor-related protein Megalin may also play a role in Hedgehog signaling in vertebrates, perhaps by binding to Hedgehog proteins and facilitating their endocytosis [ 18 , 19 ]. Hedgehog pathway function in zebrafish has been analyzed primarily in the context of skeletal muscle development and differentiation [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. In zebrafish embryos at 24 h post fertilization (hpf), skeletal muscle can be subdivided into two distinct classes based on morphological characteristics and gene expression. Slow muscle fibers are mononucleate, express characteristic slow muscle forms of myosin heavy chain, and show strong nuclear expression of the transcription factor prox1 . In contrast, fast muscle fibers can be identified via their multinucleate morphology and lack of prox1 expression [ 28 ]. Cell labeling experiments have demonstrated that slow muscle fibers derive from the adaxial cells that lie immediately adjacent to the notochord [ 21 ]. As development progresses, a subset of these developing slow muscle cells migrates laterally through the myotome to form the superficial slow fibers [ 21 , 27 , 28 ]. Slow muscle fibers that remain near the midline—the muscle pioneers—express high levels of Engrailed and organize the somites into their distinctive chevron shape [ 22 , 28 , 29 , 30 ]. The remaining muscle cells in the interior of the myotome form multinucleate fast muscle fibers [ 21 , 28 ]. Many lines of evidence indicate that Hedgehog signals from axial tissues specify slow muscle in zebrafish. Slow muscle fibers are reduced or absent in embryos with Hedgehog pathway mutations [ 25 , 26 , 31 , 32 , 33 ]. Conversely, slow muscle is expanded at the expense of fast muscle in embryos with increased Hedgehog pathway activity [ 20 , 23 , 24 , 34 ]. Moreover, addition of Hedgehog protein to cultured zebrafish myoblasts induces expression of slow-muscle-specific forms of myosin heavy chain [ 35 ]. Genetic screens have identified a number of mutations disrupting the Hedgehog pathway in zebrafish [ 32 , 33 , 36 , 37 , 38 , 39 , 40 , 41 ]. Many of these Hedgehog pathway mutants share characteristic defects, the most obvious of which is abnormal somite morphology resulting from disrupted slow muscle specification and the lack of horizontal myoseptum [ 22 ]. These mutants are thus termed “ you -class” mutants because of their U-shaped somites. Five of the seven you -class mutations have been cloned, and four of these genes, syu/shh, yot/gli2, smu/smoh, and con/disp1, encode members of the Hedgehog signaling pathway [ 32 , 33 , 36 , 37 , 39 ]. The exception is ubo/prdm1, which encodes a transcriptional switch that acts downstream of Hedgehog signaling in the development of slow muscle [ 27 , 42 ]. Careful analysis reveals differences between the ubo and Hedgehog pathway mutant phenotypes. For example, Hedgehog pathway mutants have defects in the lateral floor plate of the neural tube and the dorsal aorta, which are apparently normal in ubo mutants [ 22 , 43 ]. Examination of Hedgehog-induced gene expression also reveals a clear distinction between Hedgehog pathway mutations and ubo: Hedgehog pathway mutations reduce or abolish expression of the Hedgehog target ptc1, whereas ptc1 expression is normal in ubo mutants, indicating that they can receive Hedgehog signals [ 22 , 25 , 26 , 32 , 33 , 36 , 39 ]. Previous phenotypic characterization of mutants for the eponymous you -class gene, you, has revealed delayed development of the dorsal aorta and the absence of lateral floor plate marker expression in addition to slow muscle defects [ 22 , 43 ]. Moreover, expression of Hedgehog target genes, including ptc1 and adaxial myod, is reduced in you mutants [ 25 ]. These results suggest that the you gene acts within the Hedgehog pathway itself rather than downstream of Hedgehog signaling in processes specific to slow muscle development. Prior to this study, the molecular identity of the you gene has remained unknown. We report the positional cloning of the you gene and show that it encodes a novel extracellular EGF-CUB (epidermal growth factor–complement Uegf Bmp1) protein required for Hedgehog signaling. Functional studies provide evidence that you is essential for the transport or stability of Hedgehog signals in the extracellular environment. Results you Function Is Required for Hedgehog Signaling In wild-type zebrafish embryos, the dorsal and ventral portions of each myotome converge at a point where the horizontal myoseptum forms, giving the somites their characteristic chevron shape ( Figure 1 A). In contrast, you mutants lacked the horizontal myoseptum and exhibit the U-shaped morphology that defines mutants of the you class ( Figure 1 B; [ 22 ]). The formation of the horizontal myoseptum in zebrafish depends on the proper development of slow muscle, a process that is defective in you- class mutants [ 22 , 31 ]. Hedgehog activity in the context of slow muscle cell specification can be assayed by analyzing Engrailed expression in the muscle pioneers and adaxial myod expression during somitogenesis. Wild-type embryos at 24 hpf exhibit strong Engrailed staining in characteristic elongate nuclei of 2–6 muscle pioneers—slow muscle cells that develop along the prospective horizontal myoseptum—per myotomal segment ( Figure 1 C). In addition, weaker Engrailed expression can be observed in the typically more rounded nuclei of mulitnucleate fast muscle fibers, which are situated farther from the horizontal myoseptum ( Figure 1 C; [ 28 , 30 ]). you mutant embryos completely lacked the strong Engrailed expression in the muscle pioneers ( Figure 1 D; [ 22 ]), but very weak labeling was sometimes observed in a small number of cells. When weakly expressing cells were present, they were confined to the nine most anterior somites. In wild-type embryos, myod is expressed during somitogenesis in the adaxial cells of both somitic and presomitic mesoderm, and laterally along the posterior borders of developing somites ( Figure 1 E). you mutant embryos retained the lateral expression of myod, but the adaxial expression of myod was absent in the trunk and reduced in the tail bud ( Figure 1 F; [ 25 ]). In addition, expression of ptc1— both a member of the Hedgehog pathway and a sensitive transcriptional readout of Hedgehog signaling—was reduced in the adaxial cells of you mutants at 22 somites and other stages ( Figure 1 G and 1 H; data not shown; [ 25 ]). Figure 1 you Mutants Exhibit Hedgehog-Associated Defects in Slow Muscle and Ventral Spinal Cord (A and B) Lateral views of live zebrafish at 22 hpf. Wild-type embryos (A) have chevron-shaped somites and a clearly visible floor plate (arrowhead), while you mutants (B) exhibit U-shaped somites and an indistinct floor plate (arrowhead). (C and D) Lateral views of somites 8–13 in whole-mount embryos at 24 hpf. Wild-type embryos (C) show strong Engrailed expression in muscle pioneers (arrow), and weaker expression in multinucleate medial fast fibers (arrowheads). Engrailed expression in you mutants (D) is mostly absent, though very weak expression can occasionally be observed (arrowhead). (E and F) Dorsal views of posterior trunk and tail bud in 12-somite embryos. Wild-type embryos (E) exhibit adaxial myod expression throughout the somitic (arrowhead) and presomitic (arrow) mesoderm, while you mutants (F) lack expression in the somitic (arrowhead) and in parts of the presomitic (arrow) mesoderm. (G and H) Lateral view of somites 9–15 in whole-mount embryos at 22 hpf. Wild-type embryos (G) exhibit strong expression of ptc1, while you mutants (H) show weaker levels of ptc1 expression. (I and J) Lateral view of spinal cord in the posterior trunk of whole-mount embryos at 24 hpf. Wild-type embryos (I) show expression of nkx2.2 in the ventral spinal cord, while in you mutants (J) this expression is strongly reduced. (K–N) Dorsal views of whole-mount embryos at bud stage (10 hpf). Expression in you mutant embryos of both ehh (L) and shh (N) is similar to that observed wild-type embryos (K and M). Anterior is to the left in all images. Genotypes of all embryos were determined by PCR after photography. In addition to these disruptions in developing somites, you mutant embryos showed defects in patterning of the central nervous system. nkx2.2, a Hedgehog-induced marker of ventral cell types in the spinal cord [ 44 ], was absent in the trunk and tail of you mutants ( Figure 1 I and 1 J). Moreover, our analysis and prior work has revealed that you embryos show delayed and weakened blood circulation in the dorsal aorta (data not shown; [ 45 ]). These results and previous phenotypic analyses support the conclusion that you gene function is required for Hedgehog signaling in development of slow muscle, ventral spinal cord, and the dorsal aorta. In zebrafish, three hedgehog genes —ehh, shh, and twhh —are expressed at the midline in early embryonic stages. To determine whether you is required for hedgehog gene transcription, we analyzed the expression of hedgehog genes in wild-type and you mutant embryos. Expression of all three hedgehog genes appeared normal in you embryos at bud stage (10 hpf; Figure 1 K– 1 N; data not shown). Positional Cloning of you As the first step toward identifying the you gene, we mapped the you mutation to a 1-cM (12 recombinants among 1,156 meioses) region of LG 7, between simple sequence length polymorphism (SSLP) markers Z11119 and Z15270 ( Figure 2 A). By comparing the position of the you mutation to zebrafish genetic maps ([ 46 , 47 ]; unpublished data), we excluded as candidate genes more than 60 zebrafish orthologs of Hedgehog pathway genes and genes known to interact with the Hedgehog pathway. We therefore adopted a positional cloning strategy to identify the gene disrupted by the you mutation. Using a marker linked with Z15270 on a contig from the Sanger Institute whole-genome shotgun assembly, we screened a pooled bacterial artificial chromosome (BAC) library and initiated a chromosome walk beginning with BAC zC172H20. After identifying polymorphisms in BAC end sequences, testing these polymorphisms on our mapping panel, and iteratively rescreening the pooled BAC library, we identified a contiguous stretch of genomic sequence spanning portions of five BACs with ends that flanked you ( Figure 2 A). Figure 2 Positional Cloning of the you Locus (A) Genetic and physical map of the you region on LG 7, showing the initial flanking SSLPs and the BACs used in the chromosome walk. The number of recombinants in 1,156 meioses is shown for the SSLP markers and for mapped BAC end sequences. (B) Diagram of BAC zC93A15, with expressed sequence tag markers BM184987 and AI722938 shown flanking the you locus. Both BM184987 and AI722938 showed one recombinant out of 6,514 meioses, and were genetically localized on opposite sides of you . (C) The genomic region of the you locus is depicted by horizontal black bars. Gaps in these bars represent fragments of genomic sequence that were not obtained in the sequencing analysis. Exons are depicted by blue rectangles, and the number of recombinants in 6,514 meioses is shown below each mapped exon. Single nucleotide polymorphisms in four exons always segregated with the you locus, and one of these exons harbored a single nucleotide lesion predicted to change a glutamine codon to a stop codon and truncate the open reading frame. To reduce the critical interval that contained you, we improved the resolution of our map by increasing our mapping panel to 6,514 meioses. By scoring sequences identified from BAC zC93A15 in key recombinants from the mapping panel, we localized the you gene to a portion of this BAC between polymorphisms identified in expressed sequence tag markers BM184987 and AI722938 ( Figure 2 B). Further sequence analysis and mapping identified other exons of the same gene as AI722938, some of which were on the opposite side of the mutation from the original AI722938 marker ( Figure 2 C). We isolated a full-length cDNA clone for this gene and used this new sequence information to identify polymorphisms in other exons. In all, high-resolution mapping identified four exons that failed to recombine with you and confirmed that the two ends of the gene flanked the you ty97 mutation. Sequence analysis of the four non-recombining exons in wild-type and you ty97 mutant genomic DNA identified a nonsense mutation that truncates the predicted protein approximately two-thirds of the way through the open reading frame. These findings, together with others described below, indicate that this gene is disrupted by the you mutation. you Is Orthologous to Scube2 The protein encoded by you comprises 1,010 amino acids, and comparison of the predicted you amino acid sequence against the protein database indicated that the you protein is highly similar to a family of proteins founded by mouse SCUBE1 (Signal sequence, CUB domain, EGF-related; [ 48 ]). Pairwise sequence comparisons between you and SCUBE family members revealed that the You protein most closely matched SCUBE2 in mouse (65% identity) and SCUBE2 in human (66% identity). The orthology of these genes was further supported by comparative mapping: the human genes SCUBE2, LMO1, STK33, and ST5 exhibited conserved synteny with orthologous genes in both mouse and zebrafish ([ 49 ]; unpublished data). SCUBE proteins are characterized by a signal peptide and by two types of conserved extracellular domains: EGF and CUB [ 50 ]. In all identified SCUBE family members, the N-terminal signal sequence is followed by nine EGF repeats, a spacer region, and a single C-terminal CUB domain [ 48 , 51 , 52 , 53 ]. Figure 3 A shows an alignment comparing the predicted you amino acid sequence with selected SCUBE proteins in mouse and human. Similarity between the You protein and mouse SCUBE2 was particularly high in the CUB domain (89% identity), the C-terminal sequence following the CUB domain (90% identity), and the EGF repeats (74% identity). A spacer region in the center of the amino acid sequence showed lower conservation (47% identity). Examination of this spacer region in the vicinity of the CUB domain revealed a repeated motif of six cysteine residues with characteristic and regular spacing, shown in yellow in Figure 3 A. Conservation in amino acid sequence was notably higher in this region (66% identical) than in the remainder of the spacer domain (33% identical). This six-cysteine repeat motif does not match the general structure of EGF repeats. The functional significance of this motif is not presently known, but it does lie within a region of SCUBE1 that is required in cell culture for secretion and cell surface expression [ 52 ]. Figure 3 SCUBE Protein Alignment and Truncation of the You Protein In (A) and (C), the signal peptide is labeled in blue, the nine EGF domains are labeled in red, and the CUB domain is labeled in green. (A) Alignment of the predicted You amino acid sequence with SCUBE proteins in mouse and human. Identical amino acids are shaded, and similar amino acids are boxed. Conserved cysteines in these domains and elsewhere in the alignment are indicated by filled circles. A conserved six-cysteine repeat motif N-terminal to the CUB domain is labeled in yellow. The location of the glutamine residue at amino acid 644 in the zebrafish protein, which is changed to a stop codon in you ty97 mutants, is boxed in bold. (B) Sequence traces from homozygous wild-type and you ty97 embryos. In you ty97 mutants, a C to T transition is predicted to change a glutamine codon (CAA) to a stop codon (TAA) and truncate the open reading frame. (C) Model of You protein domain structure. The You protein in you ty97 mutants is predicted to be truncated prior to the six-cysteine repeat motifs, the CUB domain, and the conserved C-terminus. In you ty97 mutants, a C to T transition alters the coding sequence at residue 644, changing a glutamine codon to a stop codon ( Figure 3 A and 3 B). The predicted mutant protein is truncated immediately prior to the six-cysteine repeat motif, so that these repeats, the CUB domain, and the highly conserved C-terminus are lacking ( Figure 3 C). Morpholino Phenocopy and RNA Rescue of you To confirm that this zebrafish EGF-CUB gene is disrupted in you mutants, we performed morpholino oligonucleotide (MO) injection experiments to phenocopy defects seen in you, and mRNA injection experiments to rescue the you phenotype in mutants ( Figure 4 ). All wild-type embryos injected with a MO targeting the translational start site showed reduced expression of myod ( n = 112) in the adaxial cells ( Figure 4 B), and an absence of strong Engrailed expression ( n = 70) in the muscle pioneers ( Figure 4 D). Embryos injected with a mismatch control MO did not exhibit these defects in either myod ( n = 65) or Engrailed ( n = 48) expression ( Figure 4 A and 4 C). Figure 4 MO-Induced Phenocopy of you Defects and Rescue of the you Phenotype with mRNA Injection (A, B, E, and F) Dorsal view of the posterior trunk and tail bud of 12-somite embryos. (C, D, G, and H) Lateral views of somites 8–13 in whole-mount embryos at 24 hpf. Anterior is to the left in all images. Injection at the 1–4-cell stage of 420 pg of a MO targeting the translational start site of the you mRNA (ATG MO) resulted in decreased adaxial expression of myod in the somitic (arrowhead) and presomitic (arrow) mesoderm of wild-type embryos (B). Injection of an equivalent amount of a mismatch control (mismatch MO) did not produce these defects (A). Similarly, wild-type embryos injected with 420 pg of the mismatch MO (C) exhibited strong Engrailed expression in muscle pioneers (arrow) and weaker expression in medial fast fibers (arrowheads). In contrast, Engrailed expression was strongly reduced in wild-type embryos injected with 420 pg of the ATG MO (D), though very weak expression was still observed (arrowhead). Genotypically you mutant embryos (E) showed rescued expression of adaxial myod in somitic (arrowhead) and presomitic (arrow) mesoderm when injected with 50 pg of synthetic you mRNA at the 1–4-cell stage, while mutants injected with 50 pg of a frameshift mutant form of you mRNA (F) did not exhibit rescue of adaxial myod expression. At 24 hpf, genotypically you mutant embryos injected at the 1–4-cell stage with 50 pg of you mRNA (G) showed rescue of strong Engrailed expression in the muscle pioneers (arrow) and weaker expression in the medial fast fibers (arrowheads). Mutant embryos injected with 50 pg of the mutant mRNA (H) did not show rescued Engrailed expression, though very weak Engrailed expression (arrowhead) was observed in some cases. Engrailed expression at the MHB was normal in all analyzed embryos (data not shown). Genotypes of embryos shown in (E–H) were determined by PCR after photography. When injected with 50 pg of synthetic wild-type you mRNA, 98.7% ( n = 665) of embryos from you/+ intercrosses showed expression of myod in adaxial cells at 12 somites ( Figure 4 E). Genotyping of 571 embryos with wild-type myod expression from these intercrosses showed that 137 (24%) were homozygous mutant for you . In contrast, 23.6% ( n = 127) of embryos from you/+ intercrosses injected with a mutant form of you mRNA lacked expression of myod in adaxial cells ( Figure 4 F); 20 of the mutants were genotyped and confirmed to be you mutant homozygotes. Similarly, all embryos ( n = 62) from a you/+ intercross injected with wild-type mRNA showed strong Engrailed labeling in the muscle pioneers ( Figure 4 G; genotyping of 32 phenotypically wild-type embryos showed that seven were homozygous you ty97 ), whereas 24% ( n = 33) of embryos injected with control mRNA lacked Engrailed expression ( Figure 4 H; eight phenotypic mutants were confirmed as homozygous for the you mutation). In addition, injection of you MOs resulted in loss of nkx2.2 expression in the trunk and tail of wild-type embryos at 24 hpf, and injection of 50 pg of you mRNA was sufficient to rescue nkx2.2 expression in you mutants (data not shown). you Expression you transcripts appear to be maternally deposited in zebrafish embryos ( Figure 5 A) and are distributed widely in the embryo through early gastrulation stages ( Figure 5 B). During late gastrulation, the distribution of you transcripts in the embryo began to be restricted, and at bud stage (10 hpf) you was expressed in the eye field, in distinct bilateral domains within the developing brain, and in the developing trunk of the embryo in broad paraxial stripes ( Figure 5 C). During somitogenesis, you expression continued to be refined, such that six-somite embryos exhibited expression in the eye field, in stripes in the midbrain and the midbrain–hindbrain boundary (MHB), in a complex pattern in the hindbrain, and in paraxial stripes along the anterior–posterior axis ( Figure 5 D). At 24 hpf ( Figure 5 E and 5 F), you transcripts were localized to the border of the ventral telencephalon and the dorsal diencephalon and to the ventral tectum, and were strongly expressed in the MHB, the hindbrain, and along the length of the embryo in the dorsal spinal cord. In addition, you expression was observed in the ventral tail and posterior to the yolk extension at the developing urogenital opening ( Figure 5 E). In the following 24 h of development, dorsal spinal cord expression continued, and you transcripts persisted in a complex and dynamic pattern in the brain. At 48 hpf ( Figure 5 G and 5 H), you expression was particularly strong in the cerebellum and in the hindbrain along the rhombic lip. Figure 5 Expression of you Examined by In Situ Hybridization (A and B) Maternal you transcripts were evident in cleavage-stage embryos (A) (16-cell; 1.5 hpf), and you mRNA was widely expressed into the gastrula period (B) (shield stage; 6 hpf). In addition, you mRNA was detectable by RT-PCR at 2 hpf, prior to the zebrafish midblastula transition. (C) Toward the end of gastrulation, you transcripts began to be restricted, so that at the bud stage (10 hpf), you expression was evident in the eye field (white arrowhead), in the developing midbrain and hindbrain (black arrowheads), and in posterior paraxial stripes (arrow). (D) During early somitogenesis (12 hpf), you expression was observed in the eye field (white arrowhead), in stripes in the midbrain and the MHB (black arrowheads), in a complex pattern in the hindbrain (white arrow), and in paraxial stripes along the developing trunk and tail bud (black arrow). (E and F) At 24 hpf, you transcripts were observed dorsal to the hypothalamus (black arrow), at the boundary between the telencephalon and the diencephalon (white arrow), in the ventral tectum (white arrowhead), in the region of the presumptive cerebellum (asterisk), and dorsally along the length of the spinal cord. Additional expression of you at this stage and later was observed in the ventral tail and at the urogenital opening (arrowheads; data not shown). (G and H) At 48 hpf, you transcripts were highly expressed in the cerebellum (black arrow), and were also present in the rhombic lip (white arrowhead), and continuing along the length of the anterior–posterior axis in the dorsal spinal cord (black arrowhead; data not shown). Orientation of images: (A) lateral view; (B) lateral view, dorsal to the right; (C, D, F, and G) dorsal views, anterior to the left; (E and H) lateral views, anterior to the left. Permissive Role of you Upstream of the Hedgehog Cellular Response To explore the possibility that the you gene may induce a gain-of-function phenotype when overexpressed, we injected wild-type embryos with an amount (50 pg) of synthetic you mRNA that was sufficient to rescue the phenotypic defects observed in you mutants ( Figure 6 ). When compared to embryos injected with a mutant form of you mRNA ( myod, n = 81, Figure 6 A; Engrailed, n = 25, Figure 6 E; nkx2.2, n = 17, Figure 6 I), overexpression of you in wild-type embryos did not result in obvious ectopic expression of myod ( n = 394; Figure 6 B) , Engrailed ( n = 25; Figure 6 F), or nkx2.2 ( n = 17; Figure 6 J). This result suggests that you functions as a permissive factor in Hedgehog signaling, rather than as a potent activator of the Hedgehog pathway. When 50 pg of synthetic mRNA encoding a potent Hedgehog pathway activator (shh) was injected into embryos from a you/+ intercross, ectopic expression of myod ( n = 53; Figure 6 C and 6 D), Engrailed ( n = 78; Figure 6 G and 6 H), and nkx2.2 ( n = 78; Figure 6 K and 6 L) was induced in all embryos. Genotyping a subset of these embryos indicated that both genotypically wild-type and you mutant embryos showed ectopic expression of each of these markers (myod: 41 wild-type, 12 you; Engrailed: 34 wild-type, 8 you; nkx2.2: 63 wild-type, 15 you). Because downstream targets of the Hedgehog pathway were rescued or upregulated in shh- injected you mutants, components of the Hedgehog pathway downstream of shh are most likely functional in you embryos. These results are consistent with you acting upstream of or parallel with shh in the Hedgehog pathway. Figure 6 Early Overexpression of you in Wild-type Embryos and Rescue of you Defects by shh mRNA Injection (A–D) Dorsal views of the posterior trunk and tail bud of whole-mount embryos at 12 somites (15 hpf). (E–F) Lateral views of somites 2–7 in 24-hpf embryos. (G–H) Lateral views of somites 8–13 in 24-hpf embryos. (I–L) Lateral views of 24-hpf embryos. Anterior is to the left in all images. When 50 pg of you mRNA was injected into wild-type embryos at the 1–4-cell stage, no obvious expansion of myod (B), Engrailed (F), or nkx2.2 (J) expression was observed when compared either with wild-type embryos injected with equivalent amounts of mutant mRNA (A, E, and I) or with uninjected embryos (see Figure 1 ). Muscle pioneers were counted in a subset of the embryos; there were 4.0 ± 0.8 Engrailed-expressing muscle pioneers per somite in embryos injected with the control mRNA ( n = 3 embryos, 33 somites) and 4.6 ± 1.1 muscle pioneers per somite in embryos injected with synthetic you mRNA ( n = 8 embryos, 88 somites). Injection of 50 pg of shh mRNA into embryos at the 1–4-cell stage resulted in expansion of myod, Engrailed, and nkx2.2 expression in both wild-type (C, G, and K) and you mutant (D, H, and L) embryos. s hh injection rescued adaxial expression of myod (D), muscle pioneer expression of Engrailed (H), and ventral spinal cord expression of nkx2.2 (L) in genotypically mutant you embryos (compare with Figure 1 ). Genotypes of all embryos were determined by PCR after photography. Additional evidence that You acts upstream of the Hedgehog response derived from a loss-of-function approach, in which we activated the Hedgehog pathway by knocking down patched activity with MOs ( Figure 7 ). Expression of myod in adaxial cells was rescued or expanded in all you mutant embryos that were injected with MOs targeting ptc1 ( Figure 7 E and 7 G; n = 8 mutants) or a combination of MOs targeting both ptc1 and ptc2 ( Figure 7 I and 7 K; n = 13 mutants). you mutant embryos injected with a ptc1 mismatch control MO did not exhibit rescued myod expression ( Figure 7 A and 7 C; n = 8 mutants). In similar experiments, injection of patched MOs was sufficient to rescue or expand Engrailed expression in muscle pioneers of you mutant embryos ( Figure 7 B, 7 D, 7 F, 7 H, 7 J, and 7 L; ptc1 MO, n = 11 mutants; ptc1 + ptc2 MO, n = 11 mutants). Figure 7 Knockdown of patched Function Rescues Slow Muscle Defects in you After injection of 420 pg of a mismatch control ptc1 MO, adaxial expression of myod (A) and Engrailed (B) was normal in wild-type embryos, but absent in you mutant embryos (C and D). When injected with 420 pg of a MO targeting ptc1, however, myod expression in mutants (E) was rescued to levels comparable to wild-type embryos (G). Engrailed expression was slightly expanded in both wild-type (F) and mutant (H) embryos injected with 420 pg of ptc1 MOs. Both adaxial myod expression and Engrailed expression was slightly expanded in wild-type (I and J) and you mutant embryos (K and L) injected with MOs targeting both ptc1 and ptc2 (420 pg each). Embryos assayed for myod expression are shown in flat mount at the 12-somite stage, and somites 5–9 of Engrailed-expressing embryos are shown in lateral view at 24 hpf. Anterior is to the left in all panels. Genotypes of all embryos were determined by PCR after photography. you Acts Non-Autonomously in Muscle Pioneer Differentiation To determine whether a cell must be wild-type for you function to respond to Hedgehog signaling, we created genetic chimeras by transplanting cells from mutant embryos into wild-type hosts ( Figure 8 ). Cells derived from you mutant embryos were able to differentiate as muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei of mononucleate cells at the proper position in the somite ( Figure 8 C– 8 E; n = 13 embryos). Similarly, cells from embryos in which you function had been reduced with MOs were able to differentiate as Engrailed-expressing muscle pioneers when introduced into embryos treated with mismatch control MOs (data not shown). Figure 8 you Acts Non-Autonomously in Muscle Pioneers and Is Not Required in Cells Producing Hedgehog Signals (A) Donor embryos were labeled at the 1–4-cell stage with Oregon Green dextran. (B) Cells from donor embryos were transplanted into unlabeled hosts during late blastula and early gastrula stages. (C–E) Images from a chimera made by transplanting cells from labeled mutant donors (green in C and E) into unlabeled wild-type hosts. At 24 hpf, muscle pioneer cells in chimeric embryos were labeled with anti-Engrailed antibody (red in D and E). When transplanted into wild-type embryos, mutant cells were able to differentiate as muscle pioneers, as shown by co-labeling with the anti-Engrailed antibody (E, yellow arrows). (F–K) Images from chimeras made by transplanting cells from labeled wild-type donors (green in F, H, I, and K) into unlabeled mutant hosts. Expression of Engrailed (red in G, H, J, and K) in some mutant muscle pioneers (one marked by red arrows in G, H, J, and K) was rescued in a subset of embryos (see also Table 1 ). Donor cells in the embryo shown in (F–H) contributed solely to muscle and to non-floor-plate identities within the neural tube. Moreover, in a subset of chimeras, cells derived from wild-type donors differentiated as muscle pioneer cells (yellow arrows in J and K), simultaneously showing both the characteristic strong nuclear Engrailed expression and the typical flattened and mononucleate morphology of this cell type. The somite labeled with the arrows in J and K contains two muscle pioneers, one derived from the wild-type donor (yellow arrow) and another derived from the mutant host (red arrow). Donor cells in the embryo shown in (I–K) contributed primarily to muscle and to non-floor-plate identities within the neural tube; in addition, a group of seven floor plate cells derived from the wild-type donor was present in the tail of this embryo (not shown). Table 1 Chimeras with Wild-Type Donor Cells in you Mutant Hosts Rescue of muscle pioneers in wild-type→ you mutant chimeras was evidenced by the presence of mutant (i.e., host) cells with strong Engrailed expression in elongate nuclei at the proper position in 1–8 somites. Wild-type contribution indicates structures that included cells from labeled wild-type donors CNS, non-floor-plate cells in neural tube; FP, floor plate Muscle Pioneer Differentiation Does Not Require you Function in Axial Hedgehog-Producing Cells To determine which cell types must be wild-type for you function in order for target cells to appropriately respond to Hedgehog signals, we transplanted cells derived from wild-type donors into you mutant hosts. Of 91 chimeric mutant hosts, ten embryos exhibited rescue of Engrailed expression in genotypically mutant muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei at the proper position of posterior somites, where Engrailed is not normally expressed in you mutants ( Table 1 ; Figure 8 F– 8 K). In addition, wild-type cells differentiated as muscle pioneers in two of the ten chimeras with rescued mutant muscle pioneers. In these cases ( Figure 8 I– 8 K), the muscle pioneer identity of cells strongly expressing Engrailed was further confirmed by presence of the lineage tracer dye, which showed that these cells had the characteristic flattened and mononucleate morphology of muscle pioneers. In all ten embryos with rescued Engrailed expression, wild-type cells were present in the muscle and in non-floor-plate regions within the neural tube. In five of the chimeric embryos with rescued Engrailed expression, the floor plate and notochord were derived entirely from you mutant cells, indicating that you function is not required in axial Hedgehog-producing cells. Collectively, the transplantation experiments indicate that you function is not required in either the signaling or responding cells, and instead suggest that you is essential for the transport or stability of Hedgehog signals. Discussion Using a positional cloning approach, we have shown that the you gene encodes a novel EGF-CUB protein essential for Hedgehog signaling in zebrafish. High-resolution mapping indicates that the you locus is tightly linked to a zebrafish homolog of mouse Scube2. you ty97 mutants harbor a nonsense lesion that truncates the open reading frame upstream of the CUB domain and other highly conserved sequences. MO-mediated knockdown of the gene phenocopies defects observed in you mutants, and injection of wild-type mRNA into mutant embryos rescues the you phenotype. Taken together, these experiments provide compelling evidence that the you mutation disrupts this EGF-CUB gene. Biochemical studies of SCUBE family members have shown that they are extracellular, membrane-associated glycoproteins [ 52 , 53 ], but no previous work implicates these proteins in the Hedgehog signaling pathway. Although the Hedgehog pathway has been extensively studied in flies and mammals, several factors have likely obscured the connection between this Scube gene and the Hedgehog pathway. Prior to this study, no loss of function analysis had been performed on any Scube family gene; there is no known Scube gene in the fly, and mouse mutants have not been reported. Also, because overexpression of synthetic you mRNA does not significantly hyperactivate Hedgehog signaling, Scube gene function in the Hedgehog pathway would not be apparent in gain-of-function screens to identify pathway components. you mutant embryos exhibit phenotypic defects characteristic of reduced Hedgehog signaling in zebrafish, indicating that the you gene is a positively acting component of the Hedgehog pathway. Development of slow muscle is disrupted, as shown by lack of adaxial myod expression during somitogenesis and the absence of Engrailed-expressing muscle pioneer cells. Moreover, you mutants lack nkx2.2 expression in the ventral spinal cord, showing that specification of ventral neural fates is disrupted in this region of you embryos. Additionally, expression of the Hedgehog target gene ptc1 is reduced in you mutants. Analysis of the mutant phenotype, therefore, demonstrates that the you gene is essential for Hedgehog signaling in development of slow muscle and ventral spinal cord fates. Current evidence indicates that EGF-CUB proteins, including You, have extracellular functions. The you gene product and other SCUBE proteins contain a signal peptide sequence targeting the protein for secretion, as well as EGF and CUB domains characteristic of extracellular proteins [ 48 , 50 , 51 , 52 , 53 ]. The You homolog SCUBE1 is a glycosylated peripheral membrane protein when expressed in 293T cells, and is also present at low levels in the culture medium [ 52 ]. EGF and CUB domains are found together in a small but diverse group of extracellularly acting proteins, including the complement subunits C1s and C1r, the metalloproteinase Tolloid, the sea urchin extracellular matrix protein Fibropellin, the serum glycoprotein Attractin, and the scavenger receptor Cubilin. CUB domains have been implicated in mediating protein–protein interactions and may confer specificity to ligand binding; for example, specific CUB domains in Cubilin have been implicated in facilitating the binding and subsequent endocytosis of specific ligands (reviewed in [ 54 ]). Although you is essential for Hedgehog signaling, our analysis indicates that you mutant cells are able to produce and respond to Hedgehog signals. hedgehog gene expression in the embryonic midline is normal in you mutants, indicating that You acts downstream of hedgehog gene transcription (see Figure 1 ). Further evidence that you function is not required in cells generating Hedgehog signals derives from the analysis of chimeric embryos: muscle pioneers can differentiate in chimeras in which the notochord and floor plate are formed entirely from mutant cells (see Figure 8 ; Table 1 ). Conversely, you mutants can respond to Hedgehog pathway activation, mediated by either shh overexpression or disruption of patched, demonstrating that the defect in the mutants lies upstream of cellular response mechanism (see Figures 6 and 7 ). In addition, you mutant cells can respond to Hedgehog and differentiate as muscle pioneers when transplanted into a wild-type host (see Figure 8 ), indicating that you gene function is not required in cells responding to Hedgehog signals. The analysis of chimeras also demonstrates that the presence of wild-type cells in the paraxial mesoderm and neural plate is sufficient to allow you mutant cells to respond to Hedgehog signals produced from you mutant notochord and floor plate. Thus, our epistasis and transplantation experiments provide evidence that You functions in the extracellular environment in the field of responding cells, likely acting to transport or stabilize Hedgehog signals. It is possible that You interacts with components of the extracellular matrix, some of which are known to regulate the action of Hedgehog signals in other systems [ 11 , 12 , 55 , 56 ]. Another possible model is that You activates the Hedgehog pathway indirectly, by inhibiting an extracellular pathway antagonist such as Hip1 or Gas1. Our results, however, do not support such a model. Overexpression of high levels of synthetic you mRNA (up to ten times the amount required to rescue you mutants) in wild-type embryos did not result in obvious phenotypes when assayed by gross morphology or by expression of Hedgehog target genes, including adaxial myod, Engrailed in muscle pioneers, or nkx2.2 in the ventral spinal cord (see Figure 6 ; data not shown). The finding that overexpression of you does not significantly hyperactivate Hedgehog targets argues against simple models in which you functions to counteract an endogenous repressor of the Hedgehog pathway. An interesting aspect of the you mutant phenotype is that it encompasses only a subset of defects seen in other zebrafish Hedgehog pathway mutants. Whereas mutants for syu/shh, yot/gli2, smu/smoh, and con/disp1 have prominent midline abnormalities in the head, such as ipsilateral retinotectal projections, reduction of anterior pituitary, and defects in medial neurocranial cartilage, these phenotypes are not evident in you mutants [ 26 , 32 , 33 , 37 , 43 , 57 , 58 , 59 ]. Also, development of pectoral fins, which is disrupted in syu, con, and smu [ 26 , 60 ], appears normal in you embryos. you mutants, therefore, show characteristic Hedgehog signaling defects in slow muscle specification, patterning of ventral spinal cord, and the development of the dorsal aorta, but you is apparently not required for Hedgehog signaling in some other regions of the zebrafish embryo. Because the primary cell types disrupted in you mutants all develop in close proximity to the notochord, it is possible that you gene function may be required for the transport or stability of Hedgehog signals in the vicinity of the developing notochord but not some other regions. The notochord is a defining feature of chordates, and a notochord-associated function would explain why no you counterpart is required for Hedgehog signaling in the fly. It is not clear, however, why Hedgehog signaling near the notochord would require a special extracellular mediator. Another possibility is that maternal you function could mask earlier requirements in zygotic you mutants; future work with maternal-zygotic you mutants is needed to address this possibility. A third explanation of the requirement for you in only a subset of Hedgehog-regulated processes is that additional factors with redundant functions may substitute for you in other regions of the embryo. Intriguingly, expression of another Scube gene in mouse— Scube1— is observed in many embryonic tissues known to require Hedgehog signaling for their proper development, including the ventral forebrain, limb bud, somites, and developing gonad [ 48 ]. These results suggest that an additional zebrafish Scube gene may also play a role in the development of other areas of the embryo where Hedgehog signaling is active. Moreover, interactions between SCUBE proteins may be important for Hedgehog signaling; biochemical analysis suggests that SCUBE1 and SCUBE2 proteins can interact to form both homodimers and heterodimers [ 52 ]. In addition to its role in promoting Hedgehog signaling in the developing muscle pioneers, ventral neural tube, and dorsal aorta, the expression pattern of you suggests that the gene may act in other cell types and perhaps in other pathways. During gastrulation, when Hedgehog signaling is required for specification of muscle pioneers [ 28 ], you is widely expressed. In 24-hpf embryos, however, you is expressed strongly in specific regions in the forebrain, midbrain, and hindbrain, and dorsally along the length of the spinal cord. Some of these expression domains overlap with regions of Hedgehog signaling, whereas others do not. One region where Hedgehog activity and you expression intersect at later embryonic stages is the cerebellum, where Hedgehog signaling plays a well-defined role in the proliferation of granule cell precursors in mammals [ 61 , 62 ]. In the trunk and tail, however, you expression in the dorsal spinal cord corresponds neither with known sources of Hedgehog signals nor with cells that require Hedgehog signaling for their proper development. This result suggests that you may function in other pathways later in development. Future studies will define the role of you in the Hedgehog pathway and address the possibility that you and other Scube family genes also function in other signaling pathways. Materials and Methods Fish strains Zebrafish embryos were maintained at 28.5 °C and were staged according to [ 63 ]. Wild-type embryos were derived from the WIK strain. All phenotypic analysis of you mutants was performed with embryos homozygous for the you ty97 allele [ 22 ]. Genetic mapping The mapping panel was generated by crossing you ty97 /+ individuals with wild-type fish from the WIK strain. you ty97 heterozygotes in the F1 generation were intercrossed, and mutant and wild-type embryos in the F2 progeny were collected at 3–4 dpf for mapping. Genomic DNA was prepared from these embryos as described [ 64 ]. Primer sequences for SSLP markers were obtained from the MGH zebrafish database ( http://zebrafish.mgh.harvard.edu ). For initial localization of you, bulked segregant analysis (reviewed in [ 65 ]) was performed on DNA pools from 20 mutant and 20 wild-type embryos. Putative zebrafish orthologs of hedgehog -related genes were identified by reciprocal BLAST analysis and localized to the Heat Shock Panel as previously described [ 47 ]. BAC screening, chromosome walking, and BAC sequencing The CHORI211 BAC library was screened by PCR to identify positive BAC clones ( http://www.rzpd.de ). BAC end sequences were obtained from the Sanger Institute database ( http://trace.ensembl.org , and PCR primers were designed to amplify regions of these sequences. PCR amplicons were sequenced from homozygous wild-type and mutant embryos to identify nucleotide differences that generated restriction enzyme fragment length polymorphisms. These polymorphisms were tested on the mapping panel, and markers showing tighter linkage with you were iteratively screened against the BAC library until a contiguous stretch of genomic sequence with ends that flanked you was identified. The BAC zC93A15 was subcloned into pBluescript SK+ (Stratagene, La Jolla, California, United States) following double digest with either Pst I and EcoR I or Xba I and Xho I. Sequences were analyzed on a 3730 DNA Analyzer (Applied Biosystems, Foster City, California, United States). Sequences generated from this BAC were used in iterated searches against the zebrafish whole-genome shotgun assembly to nucleate contigs of genomic sequence. Sequencing primers were designed from these contigs and used to generate additional sequence data for zC93A15. Plasmid constructs A full-length you clone in pBluescript SK− (Stratagene) was isolated from a 15–19-hpf cDNA library (gift of Bruce Appel and Judith Eisen). A cDNA clone harboring a frameshift mutation that is predicted to truncate the you protein at amino acid residue 34 was isolated from the same library and was used as a control in overexpression experiments. A modified version of the pCS2+ expression vector was generated by cloning a 41-bp fragment into its EcoR I and Xba I sites. This stuffer fragment abolished the endogenous EcoR I, Stu I, Xho I, and Xba I restriction digest sites of pCS2+, and introduced Xba I, Sac I, Apa I, Pst I, and Xho I recognition sequences in a 5′ to 3′ orientation with respect to the SP6 promoter. Wild-type and mutant you clones were subcloned into the Xba I and Xho I sites of this modified pCS2+ vector. In situ probes for you were generated by linearizing this vector with Xba I, followed by antisense RNA synthesis with T3 polymerase. Synthetic you mRNA for injections was generated by digestion with Not I, followed by transcription using the SP6 mMessage mMachine kit (Ambion, Austin, Texas, United States). In situ hybridization, antibody labeling, and genotyping Probe synthesis, in situ hybridizations, and immunohistochemistry were performed using standard protocols. Embryos from you /+ intercrosses were genotyped after in situ hybridization and antibody labeling as described [ 66 ]. Other probes used were zebrafish myod [ 67 ], ptc1 [ 68 ], nkx2.2 [ 44 ], ehh [ 20 ], shh [ 69 ], and monoclonal antibody 4D9 [ 29 ]. Genotyping was performed by scoring a polymorphism in AI722938 (primer 1,
GTGAAAGCAAAAAGCAAGCA; primer 2,
GCACTGCATTATGTTTGTGGA; followed by a Hinf I digest). Microinjections Embryos were injected through their chorions with 500 pl of solution at the 1–4-cell stage as described [ 70 ]. RNA was diluted in 0.2 M KCl with 5 mg/ml Phenol Red prior to injection. MOs were obtained from Gene Tools (Philomath, Oregon, United States). A MO targeted to the you translational initiation site (5′-
GCCGTACAGTCCAAACAGCTCCCAT-3′) or a 5-bp mismatch control MO (5′-GCCcTAg
AGTCg
AAACAcCTg
CCAT-3′) was diluted in a 1x Danieau's solution with Phenol Red at 5 mg/ml prior to microinjection. Sequences for MOs targeting ptc1 and ptc2 were obtained from [ 28 ]. Transplantations Cellular transplantations were done according to standard methods [ 22 ]. Embryos derived from you/+ intercrosses were injected at the 1–4-cell stage with a 1% solution of Oregon Green 488 dextran (Molecular Probes, Eugene, Oregon, United States). Approximately 50–100 cells were removed from labeled donors in late blastula and early gastrula periods (4–5.3 hpf) and transplanted near the margin of unlabeled sibling hosts. Labeled donor embryos were allowed to develop until 24 hpf. Genotypes of donor embryos derived from you /+ intercrosses were determined by PCR. Genotypes of host embryos were determined by staining with Engrailed antibody. Donor cells in chimeras with wild-type cells transplanted into you mutant hosts were obtained either from WIK intercrosses or from genotypically wild-type embryos in you /+ intercrosses. Supporting Information Accession Numbers The you cDNA sequence has been deposited in GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) under accession number AY741664. The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) accession numbers for the genes and gene products discussed in this paper are Attractin (LocusID 8455), C1r (LocusID 715), C1s (LocusID 716), con/disp1 (LocusID 378448), Cubilin (LocusID 8029), ehh (LocusID 30299), Engrailed (LocusID 30244), Gas1 (LocusID 14451), Hedgehog (LocusID 42737), Hip1 (LocusID 15245), Megalin (LocusID 14725), myod (LocusID 30513) , nkx2.2 (LocusID 30697), Patched (LocusID 35851), prox1 (LocusID 30679), ptc1 (LocusID 30181), ptc2 (LocusID 30189), sea urchin Fibropellin (LocusID 373313), smu/smoh (LocusID 30225), syu/ssh (LocusID 30269), Tolloid (LocusID 42945), tout velu (LocusID 36614), twhh (LocusID 30444), ubo/prdm1 (LocusID 323473), yot/gli2 (LocusID 30154), human LMO1 (LocusID 4004) , human SCUBE1 (LocusID 80274), human SCUBE2 (LocusID 57758), human ST5 (LocusID 6764), human STK33 (LocusID 65975) , mouse SCUBE1 (LocusID 64706), and mouse SCUBE2 (LocusID 56788). | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544551.xml |
544592 | In vivo dose-response of insects to Hz-2V infection | Background Hz-2V infection of female Helicoverpa zea moths is manifested as insects that are either sterile "agonadal" individuals with malformed reproductive tissues or fertile asymptomatic carriers which are capable of transmitting virus on to their progeny. Virus infected progeny arising from eggs laid by asymptomatic carrier females may themselves be either sterile agonadals or asymptomatic carriers. Results By injecting virus into female moths, a correlation was established between virus doses administered to the females and the levels of resulting asymptomatic and sterile progeny. Conclusions The results of these experiments indicate that high virus doses produced a higher level of agonadal progeny and lower doses produced higher levels of asymptomatic carriers. | Background The insect virus, Hz-2V originally named gonad-specific virus (GSV) [ 1 ] was first identified in moths from a colony of corn earworms, Helicoverpa zea originating at the USDA-ARS in Stoneville, MS [ 1 , 2 ]. Insects infected with this virus were found to have malformed and missing reproductive tissues and were sterile, a condition that has been referred to as "agonadal". The examination of infected moths revealed that this virus replicated in a variety of male and female reproductive tissues including the common and lateral oviducts. Hence the tropism and replication of the virus is not specific to gonadal tissues. This rod shaped, enveloped, DNA virus has been more appropriately named Hz-2V since it resembles Hz-1V in size, pathology in vitro and in genome structure and size [ 3 - 5 ]. While examining progeny from eggs laid by infected female moths, Hamm et al . [ 2 ] identified individuals that appeared healthy and were capable of transmitting Hz-2V to their progeny. Using PCR analysis Lupiani et al . [ 6 ], were able to detect viral DNA sequences in feral corn earworms from wild populations that appeared healthy. These apparently healthy, infected moths are asymptomatic carriers of Hz-2V. The ability of this virus to persist in these asymptomatic carriers is a key feature of the biology of this virus. Since productive replication of Hz-2V results in the gross malformation of reproductive tissues and sterility of infected adult moths, persistence in asymptomatic carrier moths allows the virus to be maintained in insect populations such as the Stoneville colony. Hamm et al . [ 2 ] presented evidence from experimental matings involving asymptomatic female moths and uninfected males that showed the proportion of agonadal progeny arising from eggs laid on successive oviposition days increased rapidly with each oviposition day, suggesting a change in viral activity in the asymptomatic female. They proposed that the outcome of virus infection in progeny was related to virus dose, such that eggs laid on early oviposition days received a low virus dose resulting in more asymptomatic virus carrier moths, whereas those arising from later oviposition days received a high virus dose and developed into agonadal moths. These findings indicate that Hz-2V is able to exist in a persistent or latent state in some corn earworms and become induced into productive replication at a specific time in the development of the insect. During their experiments, Hamm et al . [ 2 ] were unable to accurately determine and control the virus dose female moths received and they were unable to directly detect females that were asymptomatic carriers of the virus. Raina et al . [ 7 ] showed that it was possible to inject Hz-2V into healthy female corn earworm moths, and upon mating with healthy male moths, produce asymptomatic carrier and agonadal progeny. They found that about half of all of the progeny produced by females that were infected with a moderate virus dose exhibited the agonadal condition and that about 90% of the remaining apparently healthy progeny actually carried viral DNA sequences detectable by PCR. This data suggests that adult females can be injected with virus to experimentally produce females that mimic the asymptomatic carrier females described by Hamm et al . [ 2 ]. In this study we have used the approach of injecting virus into healthy female moths to examine the relationship between virus dose and the level of infected, agonadal and asymptomatic carrier progeny insects hatching from eggs laid on successive ovipostion days. The results presented here demonstrate that virus dose affects both the level of infected progeny and the kind of infection found in insects hatching from eggs laid by virus infected females, indicating a direct correlation between virus dose received by females and the level of infected progeny they produce. Also demonstrated here is the fact that for each virus dose, as the level of agonadal insects hatching from eggs laid on successive oviposition days increase, the level of asymptomatic carrier progeny decreases. Results A total of 1856 progeny moths resulting from approximately116 eggs laid on each of the first four oviposition days by females infected with 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , or 2 × 10 8 TCID 50 units of Hz-2V were dissected and the reproductive tissues examined for signs of virus pathology. The PCR products of DNA samples from reproductive tissues of all apparently healthy progeny moths were examined for the presence of Hz-2V DNA via slot blot hybridization (figure 1 ), and the size of the PCR products of representative samples was determined by agarose gel electrophoresis. The results of agarose gel electrophoresis of PCR products from representative samples of agonadal, asymptomatic carriers and apparently healthy moths are shown in figure 2 . Figure 1 Slot blot hybridization results of DNA extracted from reproductive tissues of corn earworm moths. DNA was extracted, amplified via PCR, transferred onto a nylon membrane, and hybridized with a DIG-labeled viral DNA probe. Dark blots are indicative of DNA from asymptomatic carrier moths (As). Blots of DNA samples from insects from the healthy colony (H) and from insects that were determined to be apparently healthy (Ah) were blank or very light. Figure 2 Agarose gel of PCR products from DNA extracted from the reproductive tissues of corn earworm moths. The first lane is from a sample containing purified Hz-2V DNA (V). Lanes 2 denotes a sample from normal, healthy moth (H) from our insect colony. Lane 3 contains a DNA sample from an apparently healthy (AH) progeny moth arising from and infected female. Lanes 4–8 contain DNA samples extracted from asymptomatic progeny corn earworm moths (AS). Moths that had reproductive tissues that appeared to be normal but tested positive for Hz-2V DNA by PCR analyses were considered asymptomatic carriers of the virus. For each virus dose tested the number of agonadal moths, asymptomatic carriers, infected individuals (the sum of agonadal and asymptomatic carriers), and uninfected progeny moths hatching from eggs laid on each oviposition day was recorded. The analysis of these results showed that the percentage of total infected progeny (asymptomatic carriers and agonadal moths) at all doses tested increased with each successive oviposition day, and the level of infected progeny increased as virus dose increased from 2 × 10 5 to 2 × 10 8 TCID 50 units (figure 3 ). For individuals hatching from eggs on oviposition day one, the highest percentage of infected progeny (approximately 80%) was produced by females infected with the two highest virus dose (2 × 10 7 and 2 × 10 8 ), whereas the lowest percentage (about 60%) was produced by females infected with the lowest doses of virus (2 × 10 5 and 2 × 10 6 TCID 50 ). Figure 3 Mean percentages of infected (agonadal and asymptomatic carriers) progeny arising from eggs laid by female moths infected with 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , or 2 × 10 8 TCID 50 units of Hz-2V. Virus infected progeny moths arising from eggs laid on each oviposition day by females infected with different virus doses were divided into agonadal and asymptomatic carriers and these results are presented in figure 4 . No agonadal insects arose from eggs laid on oviposition day one by females infected at the lowest virus doses, whereas approximately 15% of the progeny females from eggs laid at this time by females infected at the two highest doses were agonadal. At all of the viruses doses tested, between 70 and 90% of the individuals hatching from oviposition day one eggs were asymptomatic carriers (figure 4 ). Figure 4 Mean percentages of all (male and female) agonadal (AG) and asymptomatic carrier (AS) F1 moths arising from eggs laid by females infected with 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , or 2 × 10 8 TCID 50 units of Hz-2V. For all F1 insects hatching from eggs laid on day two, (figure 4 ) the percentage of agonadal moths increased with increasing virus dose and the percentage of asymptomatic carriers at each dose declined (figure 4 ). The highest number of agonadal moths (approximately 70%) hatching from eggs laid on day two came from females that received the highest virus dose. At the two lowest doses the level of agonadals hatching from day two eggs was between 5 and 20%. At all doses almost 100% of the eggs laid on days three and four gave rise to agonadal moths. In order to better illustrate the relationship between the two types of infections and to emphasize the effects of virus dose upon the proportions of asymptomatic and agonadal infections, percentages of asymptomatic carriers and agonadal progeny for only the highest and lowest dose are presented in figure 5 . The trend in the two types of infected progeny insects follows the same general pattern for both virus doses relative to oviposition day. That is, at both doses the percentage of agonadal progeny increases with ovipostion day as the percentage of infected insects that are asymptomatic carriers of Hz-2V decreases. At the highest dose, the proportion of agonadal insects starts out higher (~ 10%) on the first oviposition day than that of agonadal progeny of females infected at the lowest dose (0%), and rises more quickly to ~ 70% of the progeny from eggs laid on oviposition day two. This is compared to only about 5% of the progeny arising from day two eggs laid by females infected at the lowest dose. Interestingly the reverse is the case for asymptomatic progeny hatching from oviposition day one eggs. Whereas approximately 90% of the infected oviposition day one individuals from females infected at the lowest virus does are asymptomatic only about 10% of the individuals from females infected at the highest dose are asymptomatic. Figure 5 Mean percentages of all (male and female) agonadal (AG) and asymptomatic carrier (AS) F1 moths arising from eggs laid by females infected at the highest and lowest doses of Hz-2V. Discussion Injecting Hz-2V into female moths results in experimentally infected insects that resemble asymptomatic females and females that have become infected with the virus during copulation, not unlike the females infected during mass-matings by infected males in transmission experiments conducted by Hamm et al . [ 2 ]. These infected moths appear healthy, are fertile, and can transmit the virus to progeny that result from mating. Some of the progeny moths arising from these infected females do not carry any detectable Hz-2V DNA sequences, others are sterile with malformed reproductive tissues, and still others are fertile, asymptomatic carriers of the virus. This variety of infections suggests that the virus is not initially present in all of the eggs laid by infected females, but is transmitted transovarially to some of the eggs at sometime time prior to oviposition. This idea is important in that it suggests that the dose or titer of virus transmitted from the parent female moth to the developing oocytes is not constant, and that the virus dose that each oocyte receives determines the outcome of the infection when these progeny insects mature into adult moths. The precise molecular mechanism that determines which infected individuals become agonadal and which will maintain the virus in the population as asymptomatic carriers has yet to be determined. The results presented in figure 4 , demonstrate that the percentage of agonadal progeny resulting from eggs laid on oviposition day one by female moths infected with Hz-2V increased as the dose of Hz-2V used to infect female moths increased. Progeny arising from eggs laid on oviposition day two also exhibited this correlation between virus dose and percent agonadal progeny. This indicates that the titer of the virus present in the experimentally infected female moths determines the amount of virus that is transmitted to eggs, and is directly correlated to the percentage of agonadal progeny arising from eggs laid by the infected females. As the dose of Hz-2V used to infect a female moth is increased, a corresponding increase is observed in total agonadal progeny arising from all eggs laid by the infected female. The percentage of agonadal progeny also increases with each successive oviposition day, approaching 100% agonadal progeny by day three at all virus doses tested, and all progeny moths arising from eggs laid on oviposition day four in all groups were agonadal. Based on the correlation between virus titer and percent agonadal progeny observed in these experiments, the increase in agonadal progeny per oviposition day is likely due to an increase in the titer of virus transmitted to the eggs, suggesting that the titer of virus increases in the parent female moths with each successive oviposition day. Studies of Hz-2V replication in vitro revealed a rapid increase in virus titer by 24 hours post infection in Tn-368 and Ld-652Y cells [ 4 , 5 ]. Hz-2V replication in vivo in the epithelial cells of agonadal female oviduct tissue has been described previously by Rallis and Burand [ 8 ]. The level of detectable virus in these tissues increased dramatically between 8 days post pupation (dpp), measured from the day the last larval exuviae was shed, and 10 dpp. It is likely that the large increase in virus over a 24 hour cycle observed in vitro also occurs in vivo , resulting in a significant daily increase in the titer of Hz-2V in these experimentally infected female moths. Although the precise site of virus replication in these experimentally infected females is not known, the increase in virus titer in these individuals almost certainly results in an increase in virus being transmitted to the progeny with each successive oviposition day and ultimately in the patterns of infection reported here. If, as we have proposed, low virus doses result in asymptomatic carrier moths, and high virus doses produce agonadal progeny, then asymptomatic carrier progeny would likely arise from eggs produced on the earliest oviposition days and decrease with each day, as the virus titer in the egg-laying female moth increases. In fact, the percentage of asymptomatic carrier progeny in these experiments does decrease with each successive oviposition day to 0% by day four. The percent asymptomatic carriers is highest in progeny that receive the lowest virus dose, specifically progeny from oviposition day one and progeny arising from the parent female moths that were experimentally infected with the lowest dose of virus. This is directly opposite of what is observed for agonadal progeny, which is at its highest level at the highest virus dose, specifically on the later oviposition days (days three or greater) and in progeny arising from parent female moths that were infected with the highest virus dose. Interestingly, the lowest percentage of asymptomatic carrier progeny arose from eggs laid by the group of female moths that received the highest virus dose of Hz-2V (figure. 3 ). These data suggest that the virus dose transmitted by infected female moths to their developing eggs determines whether the progeny develop the agonadal condition or become asymptomatic carriers of Hz-2V. The results presented here clearly show that there is a direct correlation between virus dose and the relative percentage of agonadal and asymptomatic progeny. That is, increasing the virus dose causes an increase in the percentage of agonadal progeny, but a decrease in the percentage of asymptomatic progeny. At the present time, it is unknown how the development of an infected individual into an agonadal adult or an asymptomatic carrier is regulated. It is likely that a minimum titer of Hz-2V is needed at a key point in larval development to produce a viral factor(s) within the larval tissues at a threshold level required to reprogram the development and differentiation of the reproductive tissues into the agonadal structures. If this threshold is equaled or exceeded at this point in development, the progeny will exhibit the agonadal condition. However, if this threshold level is not attained, then the reproductive tissues are not reprogrammed and the infected insect becomes an apparently healthy, fertile, asymptomatic carrier of Hz-2V. Conclusions The evolution of Hz-2V infection in H. zea has resulted in the ability of the virus to produce two different types of infections in the insects that enable the virus to replicate to high titers in the reprogrammed reproductive tissues in sterile agonadal moths, while maintaining itself in a population in asymptomatic carrier moths. This replication strategy appears to be essential for the continued existence of Hz-2V, since the development of the sterile, agonadal condition in all infected moths would lead to the extinction of the insect host, and the possibly the virus as well. The production of asymptomatic carrier moths ensures that some fertile, infected moths exist that can mate and produce infected progeny, enabling an Hz-2V-infected population to sustain itself, as in the case of the Stoneville colony. Methods Source of insects and virus Corn earworm larvae used to start a laboratory colony of healthy H. zea were obtained from the USDA-ARS in Stoneville, MS. Insects were reared on artificial diet and maintained as outlined previously [ 9 ]. Hz-2V for infecting female moths was prepared as described previously and purified via sucrose gradient centrifugation [ 4 ]. Injection of adults Newly emerged adult female moths were prepared and injected with Hz-2V as outlined by Rallis and Burand [ 8 ]. The female moths were divided into four dose groups, and 9 or 10 insects were infected with Hz-2V at one of the following concentrations of 2 × 10 5 , 2 × 10 6 , 2 × 10 7 , and 2 × 10 8 TCID 50 units. TCID50 assays Tn368 cells were cultured as per Burand & Lu [ 4 ] and 100 ul of cell culture medium containing 8 × 10 4 Tn368 cells were seeded into each well of a 96-well plate. Between 6 and 13 serial dilutions were made from each virus sample assayed and 10 or 20 wells were plated with 10 ul for each dilution. Plates were incubated at 27°C for 3 to 4 days and examined for the appearance of cytopathic effect (CPE). The numbers of wells with CPE were counted and the TCID 50 calculated [ 9 ]. DNA extraction and purification of viral DNA DNA was extracted from the reproductive tissues of adult moths by first homogenizing dissected tissues in 200 ul of TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA, pH 8.0) followed by a 2-minute incubation in a boiling water bath. The homogenate was then chilled on ice, after which Ribonuclease A (10 ug/ul) was added to each sample, which was then incubated at room temperature for 15 min. The samples were then clarified by centrifugation at 15,600 × g for 2 min. Viral DNA used as template for PCR reactions was extracted from purified virus using 1% SDS in TE containing 1 mg/ml Protease K as outlined by Burand and Lu [ 4 ]. PCR amplification of viral DNA sequences Two sets of primers were used to amplify Hz-2V genomic DNA to prepare a probe for use in slot blot analysis of insect reproductive tissues. The first set (P4-1, 5'-GCACGATTCGTAATGTTC-3'; and P4-2, 5'-GCACACCTATCAATCACC-3') was designed to amplify a 434 bp sequence of the Hz-2V genome [ 6 ]. PCR reactions using P4-1 and P4-2 primers were brought to a final volume of 20 ul using the Bioneer AccuPower ® PCR reagent premix kit with 1 unit of Taq DNA polymerase. Each reaction was carried out in10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl 2 and 40 mM KCl, containing 250 uM of each of the four dNTP's, with 100 pM of P4-1 forward and P4-2 reverse primers, and 10 ng of purified viral DNA as template. These primer set and reaction conditions were also used to amplify viral DNA sequences in approximately 100 ng of DNA from reproductive tissues of moths thought to be asymptomatic carriers of Hz-2V. The second set of primers (P4-3, 5'-GCTGTGCTGTACAAGTGC-3'; and P4-4, 5'-CCCTTGACGATCCCTTTTG-3') was designed to amplify a 350 bp region directly interior to that of the P4-1 and P4-2 amplified sequence. These primers were used to generate a DIG-labeled probe for Hz-2V to be used in slot blot hybridization assays. PCR reactions for production of the DIG-labeled probe were carried out in a final volume of 50 ul using the Boehringer Manheim DIG High Prime DNA Labeling and Detection Kit, with 1X concentrations of Taq Polymerase buffer (100 mM Tris-HCl pH 8.0, 500 mM KCL pH 8.3, and 25 mM MgCL 2 ), 100 pM of both P4-3 and P4-4 primers, a hexanucleotide mixture containing DIG-labeled dUTP (2 mM dATP, dCTP, dGTP, 1.3 mM dTTP, and 0.7 mM alkali labile DIG-11 dUTP pH 7.0), and 100 pM of Hz-2V genomic DNA. The DIG-labeled PCR product was purified on a 0.8% agarose gel using the Qiagen gel electrophoresis purification kit. Both PCR reactions for amplification of the viral DNA in tissue samples and for the production of the viral DNA probe consisted of 30 cycles of a DNA denaturation step at 95°C for 1 min., a primer annealing step for 1 min. at 55°C, and a 1 min. primer extension step at 72°C. Detection of a viral DNA sequence by slot blotting To prepare the DNA for slot blot analysis, 15 ul of the P4-1 and P4-2 PCR amplified DNA from insect samples was denatured by incubating with NaOH (0.4 M)/ EDTA (10 mM, pH 8.2) at 100°C for 10 min., then applied to a Hybon-N+ membrane prewashed with 500 ul 5X SSC buffer (0.6 M NaCl, 60 mM Na citrate pH 7.0) in a Manifold II slot blotter (Schleicher & Schuell). After applying the DNA, the membrane was baked at 88°C for 2 hrs under vacuum and prehybridized for 6 hrs. at 42°C in 50% formamide prehybridization buffer (5X SSC, 0.1% (w/v) N-laurylsarcosine, 1% (w/v) Na 2 -Dodecylsulfate, 2% Blocking reagent (Boehringer-Manheim), and 50% Formamide). Slot blots were hybridized with 150 ng DIG-labeled Hz-2V probe at 37°C for 12–14 hrs. Following washing, chemiluminescent detection was carried out as recommended by the DIG High Prime Labeling and Detection Kit Manual for DNA Hybridization (Boehringer Mannheim). Analysis of PCR products by agarose gel electrophoresis In order to confirm that the PCR products that hybridized to the viral DNA probe contained an amplified DNA fragment of the appropriate size (434 bp), representative samples were analyzed by electrophoresis on 0.8% agarose gels with 0.5X TBE buffer at 100 volts for approximately 1 hr, then stained with EtBr to visualize DNA bands under ultraviolet light. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CPR participated in the design of the study, carried out the work with the insects, coordinated the project and assisted in the molecular analysis and drafting of the manuscript. JPB conceived the study, designed and supervised the experimental work and drafted the manuscript. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544592.xml |
544586 | Induced abortion and effecting factors of ever married women in the Southeast Anatolian Project Region, Turkey: a cross sectional study | Background Nearly 10% of the population of Turkey lives in the Southeast Anatolian Project (SEAP) region. The population growth rate and the rate of unintended pregnancies are high and family planning services are insufficient in this region. Lifetime induced abortion rate is also high in this region. Public health problems of the SEAP region were investigated in the "SEAP Public Health Project" in 2001 and 2002. As it is one of the most important health problems of the women living in this region; induced abortion was also investigated in this project. Methods An optimumsample size representing the rural and urban area of the region (n = 1150) was chosen by the State Institute of Statistics by a sampling method proportional to size. 1126 of the area's 1150 houses have been visited and data about induced abortions have been obtained by applying a questionnaire to 1491 ever married women who live in the region. Results It has been found that 9.0% of these women who had at least one pregnancy in their life had at least one induced abortion. The lifetime induced abortion per 100 pregnancies was found to be 2.45. The primary reason given for induced abortions was "wanting no more children" (64.6%). Lifetime induced abortions were 5.3 times greater with women using a family planning method than women not using family planning methods. Lifetime induced abortions were 4.1 times greater with unemployed women than working women. Most of the women have used private doctors in order to have an induced abortion. Although 32.29% have not yet begun to use a contraceptive method after their last induced abortion, 43.75% of the women have since started to use an effective contraceptive method. 23.96% of them have begun to use an ineffective contraceptive method. Conclusions Induced abortion is still an important problem at the SEAP region. The results of the study remind us that unemployed women and women who have more than four children is our target group in the campaign against induced abortions. Most of the women use private doctors in order to have an induced abortion. Thus, priority must be given to educate private gynecologists with respect to induced abortion. After induced abortions, a qualified family planning consultant can be given to women and they can be secured to use a suitable contraceptive method. | Background Abortion is defined by World Health Organization (WHO) as a pregnancy that ends before 28 th week of gestation. Abortions are divided into two groups as 1) induced abortion and 2) spontaneous abortion. The spontaneous abortion rate increases when the maternal and natal care is insufficient. Induced abortions occur at the desire of the couple and an increase in induced abortion rate is a good indicator of insufficient family planning services. The aim of the family planning services is the prevention of unwanted pregnancies. Inadequate access to contraceptive methods, method failure caused by misuse of the methods and non-use of effective methods are the reasons of unwanted pregnancies, which lead women to induced abortion [ 1 ]. Induced abortions have been used as a family planning method for many years and become an important problem in women's health especially in developing countries. It is one of the main causes of death of women of reproductive age [ 2 ]. Induced abortions have many health disadvantages especially when performed in unsafe conditions. In a study it has been found out that abortion may be a risk factor for subsequent depression for a period of 8 years after pregnancy occurs [ 3 ]. In another study the mortality rate for induced abortion was found to be 5.3% and this accounted for 21.1% of the total maternal deaths for this period [ 4 ]. As it is seen from these studies, induced abortions have many health disadvantages for women and thus induced abortions should not be used as a family planning method. In Turkey, the Population Planning Law legalized the provision of safe abortion services within ten weeks in May 1983. As a result, the facility to terminate unwanted pregnancies in safe conditions has been provided [ 2 ]. But induced abortion rates are different in the different regions of Turkey. Lifetime induced abortion rate is 26.6% for the whole of Turkey. However, this rate differs from 17.8% to 30.9% for the different regions of Turkey [ 2 ]. Nearly 10% of the population of Turkey lives in the Southeast Anatolian Project (SEAP) region. The population growth rate and the rate of unintended pregnancies are high and family planning services are insufficient in this region. Public health problems of the SEAP region were investigated in the "SEAP Public Health Project" in 2001 and 2002. Induced abortion was one of the health problems investigated in this project. Methods The Southeast Anatolian Project (SEAP) region has a population of approximately 6 million people and nearly 10% of the population of Turkey lives in this region. The population growth rate and the rate of unintended pregnancies are high and family planning services are insufficient in this region. Public health problems of the SEAP region was investigated in the "SEAP Public Health Project" and this project was supported by the SEAP Regional Development Management of Prime Ministry Republic of Turkey and conducted by a consortium constituted by the Turkish Parasitology Association, Gaziantep University, Dicle University (in Diyarbakır province) and Harran University (in Şanlıurfa province). Induced abortion – an important problem for women – was investigated in this project in 2001 and 2002. The population of the nine provinces in the region is 6,128,973. In order to investigate the public health problems of the region such as abortion, an optimumsample size which represents the rural and urban area of the region was determined as 6900 (d = 0.03, p = 0.04, α = 0.01). This number (6900) was divided to the average number of households (approximately 6 people live in each house in the SEAP region) and the number of houses in the sample was found to be 1150. An optimum sample size representing the rural and urban area of the region was chosen by the State Institute of Statistics by a sampling method proportional to size. Questionnaires were prepared by the academic staff of public health departments of medical faculties of the two universities (Gaziantep and Dicle Universities). Three of the questionnaires were for individuals (the questionnaire of 5 year and older girls and women, the questionnaire of 5 year and older boys and men, and the questionnaire of 0–59 month's old children) and one of the questionnaires was about the house conditions. Before the study, the questionnaires were applied to houses that were not in the study sample as a pilot study and then checked. A team for questionnaire application was constituted in every province and the teams were educated about the questionnaires. These teams visited all of the houses in the sample with a public health specialist (the head) and applied the questionnaires by face-to-face interview. Data about the people living in the house were obtained by the house questionnaire. Data about the demographic features of women, fertility, and features about abortion were obtained by the questionnaire from 5 year and older girls and women. Educated nurses (all of them were women) applied the questionnaire to women by face-to-face interview in a separate room. 1126 households of the area's 1150 houses participated to the survey. Households of the 24 houses were not found at home during the study. There were 1491 ever married (married, divorced or widow) women in the 1126 houses that participated in the survey. The data were evaluated using the SPSS 5.0 and Excel programs. Chi-square, Student's t test and logistic regression analysis were used for the statistical analysis. Results There were 1491 ever married (married, divorced or widow) women in the 1126 houses that participated in the survey. 1266 (84.9%) of these women had at least one pregnancy in their life. 9.0% of the women who were ever married and who had at least one pregnancy in their life have had at least one induced abortion in the past. The rate of the women who have had two or more induced abortions was found to be 3.63%. The lifetime induced abortion rates of the women who were ever married and who had at least one pregnancy in their life according to some basic factors are shown in Table 1 . Table 1 The lifetime induced abortion rates of the women who were ever married and who had at least one pregnancy in their life according to some basic factors Number of induced abortions 0 1 ≥2 Women who have made at least one induced abortion n % n % n % n % Total Type of residence Rural 462 93,15 21 4,23 13 2,62 34 6,85 496 Urban 690 89,61 47 6,10 33 4,29 80 10,39 770 Statistical result *p < 0.05 Age groups (year) 15–19 38 100,00 0 0,00 0 0,00 0 0,00 38 20–24 143 95,33 7 4,67 0 0,00 7 4,67 150 25–29 199 93,87 11 5,19 2 0,94 13 6,13 212 30–34 145 91,77 9 5,70 4 2,53 13 8,23 158 35–39 177 87,62 12 5,94 13 6,44 25 12,38 202 40–44 99 84,62 10 8,55 8 6,84 18 15,38 117 45–49 91 82,73 10 9,09 9 8,18 19 17,27 110 50+ 260 93,19 9 3,23 10 3,58 19 6,81 279 Statistical result *p < 0,01 Education Illiteracy 753 92,85 35 4,32 23 2,84 58 7,15 811 Literacy 85 89,47 4 4,21 6 6,32 10 10,53 95 Graduated a primary school 248 86,71 23 8,04 15 5,24 38 13,29 286 Graduated a secondary school 26 89,66 1 3,45 2 6,90 3 10,34 29 Graduated a high school or higher 40 88,89 5 11.11 0 0,00 5 11.11 45 Statistical result *p < 0.05 Employment Unemployed 567 86,17 54 8,21 37 5,62 91 13,83 658 Employed 585 96,22 14 2,30 9 1,48 23 3,78 608 Statistical result *p < 0.0001 Ethnicity Turkish 398 84,65 39 8,32 33 7,04 72 15,35 470 Kurdish 629 94,16 28 4,19 11 1,65 39 5,84 668 Arabic 93 100,00 0 0,00 0 0,00 0 0,00 93 Zaza 32 91,43 1 2,86 2 5,71 3 8,57 35 Statistical result *p < 0.0001 TOTAL 1152 91,00 68 5,37 46 3,63 114 9,00 1266 *One and two or more induced abortions have been evaluated together in the statistical analyses. The percentage of women who have made at least one lifetime induced abortion was higher in women living in urban areas (10.39%) than women living in rural areas (6.85%, p < 0.05). The percentage of lifetime induced abortion was higher in 35–49 age group (especially in 45–49 age group) than the other age groups (p < 0.01). Lifetime induced abortion rate was 7.15% in illiterate women and 10.53% in literate women and was higher among women who graduated from a primary school or higher (%12.77, p < 0.05). The percentage of women who have had at least one induced abortion was found to be higher in unemployed women and Turkish women than the other groups (p < 0.0001, Table 1 ). The lifetime induced abortion rates of the women who were ever married and who had at least one pregnancy in their life according to some fertility characteristics are shown in Table 2 . The age of the women at her first birth, number of still birth and spontaneous abortion did not affect the rate of lifetime induced abortion. The number of living children of the women was related to the number of lifetime induced abortion. The induced abortion rate was significantly high in women having 4 or more children (p < 0.01). A similar relationship was found between induced abortion and total number of pregnancies. Lifetime induced abortion was 12.09% among women who had five and more pregnancies and this was higher than the other groups (p < 0.001). Table 2 The lifetime induced abortion rates of the women who were ever married and who had at least one pregnancy in their life according to some fertility characteristics Number of induced abortions 0 1 ≥2 Women who have made at least one induced abortion n % n % n % n % Total The age of women at her first birth 12–19 770 90,06 49 5,73 36 4,21 85 9,94 855 20–24 299 91,72 17 5,21 10 3,07 27 8,28 326 25–29 43 97,73 1 2,27 0 0,00 1 2,27 44 30+ 13 100,00 0 0,00 0 0,00 0 0,00 13 Statistical result *p > 0.05 Number of still births 0 1068 91,20 61 5,21 42 3,59 103 8,80 1171 1 64 86,49 6 8,11 4 5,41 10 13,51 74 2+ 19 95,00 1 5,00 0 0,00 1 5,00 20 Statistical result *p > 0.05 Number of spontaneous abortion 0 753 90,07 47 5,62 36 4,31 83 9,93 836 1 220 91,67 15 6,25 5 2,08 20 8,33 240 2+ 179 94,21 6 3,16 5 2,63 11 5,79 190 Statistical result *p > 0.05 Number of living children 0 32 100,00 0 0,00 0 0,00 0 0,00 32 1 118 100,00 0 0,00 0 0,00 0 0,00 118 2 147 90,18 10 6,13 6 3,68 16 9,82 163 3 173 92,02 11 5,85 4 2,13 15 7,98 188 4+ 682 89,15 47 6,14 36 4,70 83 10,85 765 Statistical result *p < 0.001 Number of total pregnancies 1 96 100,00 0 0,00 0 0,00 0 0,00 96 2 120 100,00 0 0,00 0 0,00 0 0,00 120 3 127 93,38 9 6,62 0 0,00 9 6,62 136 4 104 92,86 6 5,36 2 1,79 8 7,14 112 5+ 705 87,91 53 6,61 44 5,49 97 12,09 802 Statistical result *p < 0.001 TOTAL 1152 91,00 68 5,37 46 3,63 114 9,00 1266 *One and two or more induced abortions have been evaluated together in the statistical analyses. Lifetime induced abortion was found to be significantly higher in women who had got pregnant with their last child without the desire of both of the couples, who wanted no more children and who were using a family planning method (13.31%, 11.95% and 15.21% respectively) (Table 3 ). Table 3 The number of lifetime induced abortions of women who were ever married and who had at least one pregnancy in their life according to some factors related with family planning Number of induced abortions 0 1 ≥2 Women who have made at least one induced abortion n % n % n % n % Total** Last pregnancy Wanted by both of the couples 619 92,39 34 5,07 17 2,54 51 7,61 670 Wanted by only one of the couples 139 91,45 7 4,61 6 3,95 13 8,55 152 Not wanted by both of the couples 306 86,69 25 7,08 22 6,23 47 13,31 353 Total 1064 90,55 66 5,62 45 3,83 111 9,45 1175 Statistical result *p < 0,05 The state of wanting another child Wants no more children 707 88,04 55 6,84 41 5,10 96 11,95 803 Wants immediately, wants in the future, undecided 351 95,90 11 3,00 4 1,09 15 4,09 366 Total 1058 90,50 66 5,64 45 3,84 111 9,49 1169 Statistical result *p < 0.0001 Are you using a family planning method No 596 95,97 17 2,74 8 1,29 25 4,03 621 Yes 485 84,79 49 8,56 38 6,64 87 15,21 572 Total 1081 90,61 66 5,53 46 3,86 112 9,39 1193 Statistical result *p < 0.0001 *One and two or more induced abortions have been evaluated together in the statistical analyses. **The evaluations include the ones who have answered the questions. The lifetime induced abortion rates have been evaluated considering all of the factors thought to be related with induced abortion and has been shown in Tables 1 , 2 , 3 . When we evaluate the results of logistic regression analysis; the number of total pregnancies has been found to be the factor mostly affecting the lifetime induced abortion status (Table 4 ). Every one point increase of the total number of pregnancies increases the risk of making induced abortion by 1.17 times. The family planning method usage status of the women and the employment status of the women were the other two variables affecting the lifetime induced abortion status of the women. The risk of lifetime induced abortion was found to be 5.4 times greater with women using a family planning method than women not using family planning methods. The lifetime induced abortion risk was found to be 4.1 times greater with unemployed women than working women. Table 4 The results of logistic regression Independent Variables Induced Abortion p Odds Ratio Confidence Interval (95%) Number of total pregnancies 0,0000 1,17 1,10–1,24 Family planning method Not using 1 1 Using 0,0000 5,35 3,25–8,81 Employment Employed 1 1 Unemployed 0,0000 4,12 2,51–6,77 The rate of induced abortions per 100 lifetime pregnancies – one of the most common indicators of induced abortions – was found to be 2.45. This rate is 1.38 at the rural areas and it rises to 3.33 at the urban areas (p < 0.05) (Table 5 ). Table 5 The induced abortion rate per 100 lifetime pregnancies among ever married women The induced abortion rate per 100 lifetime pregnancies Type of residence Rural 1,38 p < 0,05 Urban 3,33 Total 2,45 "Wanting no more children" is the primary reason given for lifetime induced abortion (64.58%). In 63.54% of the lifetime induced abortions both of the couples have decided to the induced abortion together. Most of the lifetime induced abortions take place at the private doctors' consultant room (46.88%) (Table 6 ). Table 6 Some characteristics of the women's last induced abortion Rural Urban Total n % n % n % The reason of the last induced abortion Wanting no more children 19 65,52 43 64,18 62 64,58 Short interval between the last two pregnancies 0 0,00 12 17,91 12 12,50 Mother's health 7 24,14 4 5,97 11 11,46 Children's health 3 10,34 3 4,48 6 6,25 The health of mother and children 0 0,00 3 4,48 3 3,13 Other 0 0,00 2 2,99 2 2,08 Who decided to the last induced abortion Both of the couples together 19 65,52 42 62,69 61 63,54 Women 2 6,90 18 26,87 20 20,83 Doctor 7 24,14 5 7,46 12 12,50 Men 1 3,45 2 2,99 3 3,13 Where did the last induced abortion take place Private doctor 15 51,72 30 44,78 45 46,88 Public hospital 9 31,03 17 25,37 26 27,08 Maternity hospital 3 10,34 4 5,97 7 7,29 Home 1 3,45 6 8,96 7 7,29 Private hospital/private polyclinic 1 3,45 5 7,46 6 6,25 Social Insurance Association 0 0,0 4 5,97 4 4,16 Mother and child health centers 0 0,0 1 1,49 1 1,04 Total 29 100,0 67 100,0 96* 100,0 *96 women have given answer to these questions. After lifetime induced abortion, 32.29% of the women have not yet begun to use a family planning method. 43.75% of them have since started to use effective methods and 23.96% of them have begun to use ineffective methods. The usage of effective methods was higher in urban areas, while the usage of ineffective methods was higher in rural areas. Intra uterine devices (IUD) (52.38%) took the first and condom (26.19%) took the second place among the effective family planning methods. Withdrawal, with a rate of 87%, took the first sequence among the ineffective family planning methods (Table 7 ). Table 7 Usage of family planning methods after lifetime induced abortion Type of residence Women using none of the family planning methods Women using an effective family planning method Women using an ineffective family planning method Total IUD Condom Oral contraceptives Sterilization of women Total effective methods Withdrawal Other ineffective family planning methods Total ineffective family planning methods n % n n n n n % n n n % n Rural 11 33,33 5 1 2 - 8 24,24 14 - 14 42,42 33 Urban 20 31,75 17 10 5 2 34 53,97 6 3 9 14,29 63 Total 31 32,29 22 11 7 2 42 43,75 20 3 23 23,96 96 In the study, lifetime induced abortions carried out by the women were also evaluated. The number of the women who have stated that "they have tried to make an induced abortion by themselves" in the past was 64. 24 of these women were from rural areas and 40 of them were from urban areas. The women who intended to carry out an induced abortion by themselves firstly preferred to use drugs (43.8%). Lifting heavy things (35.4%) took the second place. Women who live in rural areas preferred to lift heavy things (64.3%) while women in urban areas preferred to take drugs (50.0%). Discussion The percentage of having at least one induced abortion among ever married women who had at least one pregnancy in their life in the SEAP region was 9.0% (lifetime induced abortion rate). Approximately one out of ten ever married women has made at least one induced abortion in their life. Also, 2.45 induced abortion per 100 lifetime pregnancies occurred at the region. When we evaluated the results of the Turkish Demographic and Health Survey 1998; (TDHS 1998) (which is conducted to collect data on subjects such as fertility, infant and child mortality, family planning, and maternal and child health on a representative sample of Turkey through the interviews conducted with women of fertile age) the percentage of lifetime induced abortion among ever married women was reported as 18.2% and induced abortion per 100 pregnancies during the five-year period before the survey was 7.6 for the East Anatolian region (the East Anatolian and the Southeast Anatolian Regions were evaluated together as one region and the SEAP provinces take part in this region). The SEAP rates were lower than the TDHS 1998 [ 5 ]. In the TDHS 1998 the lifetime induced abortion rates of the East Anatolian Region were given. The Southeast Anatolian region provinces were evaluated in this region. This study was conducted in the Southeast Anatolian Region only. The general features and health conditions of the Southeast Anatolian Region are worse than the East Anatolian Region, explaining why the rate (9%) is lower than the TDHS 1998. There is a decrease in the lifetime induced abortion rate in the course of time compared with the TDHS 1998. Also, there is a decrease in the lifetime induced abortion rate in the same region (in the East Anatolian provinces) when the data of the TDHS 1993 is compared with the data of the TDHS 1998. Induced abortion rate per 100 pregnancies during the five-year period before the survey has decreased to 7.6 from 8.7 in the course of time [ 6 , 5 ]. A similar decrease was seen when the Turkey Reproduction Survey-1978 was compared with the TDHS 1998 [ 7 ]. In another study conducted in Turkey; abortion rate (both induced and spontaneous abortions) of ever married women was found to be 14.9% in 1991 [ 8 ]. In a resent study conducted in Manisa in 2000 induced abortion rate per 100 pregnancies during the five-year period before the survey was found to be 12.1% [ 9 ]. It is seen that the induced abortion rate is decreasing not only in the SEAP region but also in other regions of Turkey in the course of time. In a study conducted by Senlet et al. it is reported that there is a decline in induced abortion rates in Turkey [ 10 ]. However, this low lifetime induced abortion rates do not show a success because unintended pregnancies end with births in the region. As a matter of fact, 30.1% of the latest births of the women during the last five year period were not desired by both of the couples in the Southeast Anatolian region [ 11 ]. Also, total fertility rate of the women was 4.2 in the East Anatolian region [ 5 ]. The high fertility rate and the high rate of ending unintended pregnancies with births is the real cause of the low lifetime induced abortion rate in the region. The rate of induced abortion was higher in urban areas than rural areas. This was similar with the TDHS 1998 [ 5 ]. Lifetime induced abortion rate was 7.15% among illiterate women, 10.53% among literate women and was higher among women graduated from primary school or higher (% 12.77, p < 0.05). In a study conducted by Akın et al. similar results have been found [ 2 ]. Education is a very important factor effecting induced abortion rate. In the logistic regression analysis the total number of pregnancies of the women, the family planning method usage status of the women and the employment of the women have been evaluated as the independent factors affecting lifetime induced abortion. As the total number of pregnancies increases, lifetime induced abortion risk increases (odds ratio is 1.7). Women who have more than four children may be the target group of the studies planned on this subject. In a study conducted by Akın et al. a similar odds ratio (1.1) have been found [ 2 ]. Lifetime induced abortions were 4.1 times greater with unemployed women than working women. This was due to the fact that these women have lower family planning usage rates but their pregnancy rate was high. These results remind us that unemployed women and women who have more than four children must be our target group in the campaign against induced abortions as a family planning method. Lifetime induced abortions were 5.3 times greater with women using a family planning method than women not using family planning methods. I.e. the usage of family planning methods are 5.3 times higher among the women who have had an induced abortion in the past. In a study conducted by Akın et al. similar results were reported during the five-year period before the survey (odds ratio is 2.9) [ 2 ]. Lifetime induced abortions have usually taken place at a health facility and with the assistance of health personnel. After these lifetime induced abortions, a qualified family planning consultant can be appointed to these women and they can be encouraged to use a suitable contraceptive method. The rate of effective family planning method usage after induced abortion was 43.7% in our study. The same rate was 34.2% in the TDHS 1998 during the five-year period before the survey [ 5 ]. There is an increase in the rate of effective family planning method usage after lifetime induced abortion and this increase is pleasing but it is still insufficient. This increase is thought to be one of the reasons of the decrease in induced abortion rates. Similarly, Senlet and et al has reported that one of the reasons of decrease of the induced abortion rates in Turkey is due to this factor [ 10 ]. After lifetime induced abortion, 32.3% of the women were not using a family planning method in the study and this was nearly the same with the percentage evaluated in the TDHS 1998 during the five-year period before the survey (32.1%) [ 5 ]. There was no important change during the past four years. In another study in Turkey 25% of the women did not begin to use a family planning method after induced abortion [ 12 ]. In two other studies conducted in Turkey it has been found out that approximately 20% of the women did not begin to use a family planning method after induced abortion [ 13 , 14 ]. Also, 23.9% of them have begun to use an ineffective method in our study. These data shows that the family planning services are not adequate at the institutions where induced abortion is performed. Private Doctors (46.88%) and public hospitals (27.08%) were the fist two places where the women applied to have an induced abortion. Similar results have been found in the TDHS 1998 for the Eastern Anatolian provinces during the five-year period before the survey (68.4% and 19.7% respectively) [ 5 ]. Similar results were obtained in another study in our country and it has been found out that 50% of the induced abortions were made by private doctors and private doctors were the first place chosen for induced abortion [ 15 ]. Thus, priority must be given to educate private gynecologists. After lifetime induced abortion, 67.71% of the women have begun to use a family planning method in our study. The primary reason given for the last induced abortion was "wanting no more children" (64.5%) and this is similar with the data of the TDHS 1998 [ 5 ]. This is also another indicator for high unintended pregnancy rates and insufficient family planning services in the region. Similar results have been obtained in a different study in our country. In this study 47.6% of the women requested an induced abortion because they wanted no more children [ 16 ]. Although the rate of lifetime induced abortions are decreasing in the course of time it is still an important health problem in the SEAP region. Unintended pregnancy and total fertility rates of the region is still higher than the other regions of Turkey. Thus, family planning services, the educational level of women and the status of women need improvement. Conclusions Although 9.0% of the ever married women who had at least one pregnancy in their life have made at least one induced abortion and 2.45 induced abortion per 100 lifetime pregnancies occurred at the SEAP region, these rates are lower than the whole rate of Turkey. But, the high fertility rate shows us that family planning services are insufficient in the region. Also 32.29% have not begun to use a contraceptive method after their last induced abortion and 23.96% of them have begun to use an ineffective contraceptive method. This shows an important lack on this subject. After these lifetime induced abortions a qualified family planning consultant can be appointed to these women and they can be encouraged to use a suitable contraceptive method. Also to decrease lifetime induced abortions; women who have more than four children and unemployed women may be the target group of studies planned on this subject. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AİB participated in the conception and design, provision of study materials, analysis of the data, statistical expertise, drafting the article and revision of the article. BÖ participated in the conception and design, collection and assembly of data, provision of study materials, analysis of the data, statistical expertise, drafting the article, revision of the article and final approval of the article. SÖ participated in the conception and design, collection and assembly of data, provision of study materials, analysis of the data, statistical expertise, drafting the article, revision of the article and final approval of the article. SŞ participated in the conception and design, collection and assembly of data, provision of study materials, analysis of the data, statistical expertise, drafting the article, revision of the article and final approval of the article. TŞ participated in the collection and assembly of data, provision of study materials, analysis of the data and statistical expertise. GS participated in the conception and design, collection and assembly of data, provision of study materials, analysis of the data and statistical expertise. AC participated in the conception and design, collection and assembly of data, provision of study materials, analysis of the data and statistical expertise. Eİ participated in the conception and design, collection and assembly of data, provision of study materials, analysis of the data, statistical expertise, drafting the article, revision of the article and final approval of the article. HA participated in the collection and assembly of data, provision of study materials, analysis of the data and statistical expertise. YP participated in the collection and assembly of data, provision of study materials, analysis of the data and statistical expertise. FA participated in the conception and design, collection and assembly of data, provision of study materials. MA participated in the conception and design, collection and assembly of data, provision of study materials. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544586.xml |
539237 | Age-dependent plasticity in the superior temporal sulcus in deaf humans: a functional MRI study | Background Sign-language comprehension activates the auditory cortex in deaf subjects. It is not known whether this functional plasticity in the temporal cortex is age dependent. We conducted functional magnetic-resonance imaging in six deaf signers who lost their hearing before the age of 2 years, five deaf signers who were >5 years of age at the time of hearing loss and six signers with normal hearing. The task was sentence comprehension in Japanese sign language. Results The sign-comprehension tasks activated the planum temporale of both early- and late-deaf subjects, but not that of hearing signers. In early-deaf subjects, the middle superior temporal sulcus was more prominently activated than in late-deaf subjects. Conclusions As the middle superior temporal sulcus is known to respond selectively to human voices, our findings suggest that this subregion of the auditory-association cortex, when deprived of its proper input, might make a functional shift from human voice processing to visual processing in an age-dependent manner. | Background There is evidence that cross-modal plasticity induced by auditory deprivation is apparent during sign-language perception. Sign languages involve the use of the hands and face, and are perceived visually [ 1 - 3 ]. Using functional MRI (fMRI), Neville et al. [ 1 ] observed increased activity in the superior temporal sulcus (STS) during the comprehension of American Sign Language (ASL) in both congenital deaf subjects and hearing native signers. The authors therefore suggested that the STS is related to the linguistic analysis of sign language. Nishimura et al. [ 2 ] found that activity was increased in the auditory-association cortex but not the primary auditory cortex of a prelingual-deaf individual during the comprehension of Japanese sign language (JSL). After this patient received a cochlear implant, the primary auditory cortex was activated by the sound of spoken words, but the auditory association cortex was not. The authors suggested that audio-visual cross-modal plasticity is confined to the auditory-association cortex and that cognitive functions (such as sign language) might trigger functional plasticity in the under-utilized auditory-association cortex. In addition, Pettito et al. [ 3 ] observed increased activity in the superior temporal gyrus (STG) in native deaf signers compared with hearing non-signers. These findings suggest that the changes associated with audio-visual cross-modal plasticity occur in the auditory-association cortex. However, the age dependency of this plasticity is not known. To depict the age dependency of the cross-modal plasticity, we conducted a functional MRI study of deaf signers with both early and late deafness, as well as hearing signers, performing a sign-comprehension task. 'Early deaf' subjects were defined as those who lost their ability to hear before the age of 2 years, whereas 'late deaf' subjects lost their hearing after the age of 5 years. Results Performance on the JSL comprehension task was similar across the groups (F(2, 14) = 1.279, P = 0.309, one-way ANOVA). The patterns of activity evoked during the sign-comprehension task in the hearing signers and the deaf groups are shown in Figure 1 . Within the temporal cortex, all groups showed activation in the occipito-temporal junction extending to the portion of the STG posterior to the Vpc line (an imaginary vertical line in the mid-sagittal plane passing through the anterior margin of the posterior commissure). In the early- and late-deaf subjects, the activation of the posterior STG extended anteriorly to the Vpc line to reach the Vac line (an imaginary vertical line in the mid-sagittal plane passing through the posterior margin of the anterior commissure). The activation was confined to the STG, extending into the superior temporal sulcus, and was more prominent on the left side. A direct comparison between early- and late-deaf subjects revealed significantly more prominent activation of the bilateral middle STS in the early-deaf subjects (Figure 1 ). Discussion The onset of deafness is related to language acquisition. Prelingual deafness occurs before spoken language is learned. Hearing people generally learn their first language before 5 years of age; hence, prelingual deaf individuals are either deaf at birth or became deaf prior to developing the grammatical basis of their native language, which is usually before the age of 5 years. Postlingual deafness is the loss of acoustic senses, either suddenly due to an accident or as a gradual progression after native-language acquisition [ 4 ]. Hence, the early-deaf subjects in the present study are categorized as 'prelingual deaf' and the late-deaf subjects are categorized as 'postlingual deaf'. More than 90% of children with prelingual hearing loss have parents with normal hearing [ 5 ]. Furthermore, in Japan, the traditional teaching method for deaf children includes aural/oral methods, such as lipreading. Native signers are usually limited to those who were brought up by deaf parents. Because of this, the majority of prelingual deaf subjects learn spoken language (Japanese) in artificial ways, such as aural/oral methods. In the present study, the parents of the deaf subjects all had normal hearing. Five out of six of the early-deaf subjects started JSL training after the age of 6 years. Thus, JSL is not the first language for any of the groups in the present study. The posterior STS was activated in all groups during sign comprehension, which is consistent with the proposed neural substrates that subserve human movement perception [ 6 ]. The posterior STS region is adjacent to MT/V5, which is consistently activated during the perception of human body movement [ 7 - 9 ]. Hence, the activation of the posterior STS in both hearing and deaf subjects is related to the perception of the movement of the hands and mouth. Both the early- and late-deaf groups showed activation in the planum temporale, whereas hearing signers did not. Anatomically, the anterior border of the PT is the sulcus behind Heschl's gyrus and the medial border is the point where the PT fades into the insula. The posterior border of the PT involves the ascending and descending rami of the Sylvian fissure [ 10 ]. Functionally, the left PT is involved in word detection and generation, due to its ability to process rapid frequency changes [ 11 , 12 ]. The right homologue is specialized for the discrimination of melody, pitch and sound intensity [ 13 , 14 ]. It has been shown that non-linguistic visual stimuli (moving stimuli) activate the auditory cortex in deaf individuals, but not in hearing subjects [ 15 , 16 ]. McSweeney et al. [ 17 ] showed that the planum temporale is activated in deaf native signers in response to visual sign-language images and this activation is larger for native deaf signers compared to hearing signers. Our previous study [ 18 ] revealed that cross-modal activation in the temporal cortex of the deaf subjects was triggered not only by signs but also by non-linguistic biological motion (lip movement) and non-biological motion (moving dots). Signs did not activate the temporal cortex of either the hearing signers or the hearing non-signers. Thus, in the present study, the activation of the planum temporale in the early- and late-deaf subjects is probably due to the effects of auditory deprivation, rather than linguistic processes. This theory is also supported by the fact that the hearing signers in the present study did not show temporal-lobe activity during JSL comprehension, whereas the PT was more prominently activated in the deaf subjects irrespective of the timing of the onset of deafness. These findings indicate that auditory deprivation plays a significant role in mediating visual responses in the auditory cortex of deaf subjects. This is analogous with findings related to visual deprivation: irrespective of the onset of blindness, the visual-association cortex of blind subjects was activated by tactile-discrimination tasks [ 19 , 20 ] that were unrelated to learning Braille [ 20 ]. These results suggest that the processing of visual and tactile stimuli is competitively balanced in the occipital cortex. A similar competitive mechanism might occur in the PT following auditory deprivation. Activation of the STG in hearing subjects during lipreading [ 21 ] indicates which cortico-cortical circuits might be involved in the competitive balance between the modalities. In fact, we found that the cross-modal plasticity in the deaf subjects occurred within the neural substrates that are involved in lipreading in hearing subjects [ 18 ]. The middle STS, anterior to the Vpc line, was activated more prominently in the early- than the late-deaf subjects. This difference is probably not related to linguistic processes, as both early- and late-deaf subjects are equally capable of learning JSL with the same amount of training. The middle STS region is presumably the area that is selective to human voice processing [ 22 ]. This area is known to receive predominantly auditory input, being involved in the high-level analysis of complex acoustic information, such as the extraction of speaker-related cues, as well as the transmission of this information to other areas for multimodal integration and long-term memory storage [ 22 ]. This implies that early auditory deprivation (at <2 years of age) might shift the role of the middle STS from human voice processing to the processing of biological motion, such as hand and face movements (cross-modal plasticity). It has been suggested that once cross-modal plasticity occurs in the auditory cortex, the restoration of auditory function by means of cochlear implants is ineffective [ 23 ]. Hence, the first 2 years of life might be the sensitive period for the processing of human voices. Considering that the STS voice-selective area is not sensitive to speech per se but rather to vocal features that carry nonlinguistic information [ 22 ], the functional role of this region in early-deaf subjects with regard to the paralinguistic aspects of sign language is of particular interest and further investigation will be necessary. Conclusions The results of the present study suggest that in early-deaf subjects, non-auditory processing, such as that involved in the perception and comprehension of sign language, involves the under-utilized area of the cortex that is thought to be selective to the human voice (middle STS). This indicates that the sensitive period for the establishment of human voice processing in the STS might be during the first 2 years of life. Methods The subjects comprised six early-deaf signers (mean age: 22.8 ± 3.1 years), five late-deaf signers (mean age: 34.4 ± 16.2 years) and six hearing signers (mean age: 33.7 ± 12.1 years; Table 1 ). The early-deaf subjects lost their hearing before 2 years of age, whereas the late-deaf subjects became deaf after the age of 5 years. The parents of all subjects had normal hearing. None of the subjects exhibited any neurological abnormalities and all had normal MRI scans. None of the cases of deafness were due to a progressive neurological disorder. All deaf and hearing subjects were strongly right handed, except for one late-deaf subject who was ambidextrous, according to the Edinburgh handedness inventory [ 24 ]. The study protocol was approved by the Ethical Committee of Fukui University School of Medicine, Japan, and all subjects gave their written informed consent. The tasks involved the passive perception of JSL sentences that are frequently used in the deaf community. JSL, which has its own grammar, morphemes and phonemes, is different from spoken Japanese at all levels. JSL utilizes facial expressions as obligatory grammatical markers, as does ASL [ 25 ]. The fMRI session with JSL consisted of two rest and two task periods, each of 30 seconds duration, with alternating rest and task periods. During the 30-second task period, the subjects were instructed to observe a JSL sentence presented every 5 seconds by a male deaf signer in a video, which was projected onto a screen at the foot of the scanner bed and viewed through a mirror. The sentences were relatively short and straightforward; for example, "I cut a piece of paper with scissors". During the 30-second rest period, the subjects fixed their eyes on the face of a still image of the same person. Each session started with a rest period and two fMRI sessions were conducted. The procedure was identical for all hearing and deaf subjects. After the fMRI session, outside of the scanner, the subjects were presented the JSL sentences used during the session. These were shown one by one on the video screen and the subjects were required to write down the presented sentences in Japanese. On each presentation, the subjects were asked if they had seen the JSL sentence in the scanner, in order to confirm that they had been engaged in the task during the session. The percentage of correct responses was calculated as the number of correctly written sentences divided by the number of presented sentences. A time-course series of 43 volumes was produced using T2*-weighted gradient-echo EPI sequences with a 1.5 Tesla MR imager (Signa Horizon, General Electric, Milwaukee, Wisc., USA) and a standard birdcage head coil. Each volume consisted of 11 slices, with a slice thickness of 8 mm and a 1-mm gap, which covered the entire cerebral cortex. The time interval between two successive acquisitions of the same image was 3,000 ms, the echo time was 50 ms and the flip angle was 90 degrees. The field of view was 22 cm. The digital in-plane resolution was 64 × 64 pixels. For anatomical reference, T1-weighted images were also obtained for each subject. The first three volumes of each fMRI session were discarded because of unstable magnetization. The remaining 40 volumes per session were used for statistical parametric mapping (SPM99, Wellcome Department of Cognitive Neurology, London, UK) implemented in Matlab (Mathworks, Sherborn, Mass., USA) [ 26 , 27 ]. Following realignment and anatomical normalization, all images were filtered with a Gaussian kernel of 10 mm (full width at half maximum) in the x , y and z axes. Statistical analysis was conducted at two levels. First, the individual task-related activation was evaluated. Second, the summary data for each individual were incorporated into the second-level analysis using a random-effects model to make inferences at a population level. The signal was proportionally scaled by setting the whole-brain mean value to 100 arbitrary units. The signal time course for each subject was modeled using a box-car function convolved with a hemodynamic-response function and temporally high-pass filtered. Session effects were also included in the model. The explanatory variables were centered at zero. To test hypotheses about regionally-specific condition effects (that is, sentence comprehension compared with rest), estimates for each model parameter were compared using the linear contrasts. The resulting set of voxel values for each contrast constituted a statistical parametric map (SPM) of the t statistic (SPM{ t }). The weighted sum of the parameter estimates in the individual analyses constituted 'contrast' images that were used for the group analysis. Contrast images obtained via individual analyses represent the normalized task-related increment of the MR signal of each subject. To examine group differences (prelingual deaf, postlingual deaf and hearing signers) in activation due to the sign-comprehension task, a random-effect model was performed with the contrast images (1 per subject) for every voxel. Using the a priori hypothesis that there would be more prominent activation in the early- than late-deaf subjects, we focused on the temporal cortex, which was anatomically defined in standard stereotaxic space [ 28 ]. The threshold for SPM{ t } was set at P < .001 without a correction for multiple comparisons. Authors' contributions NS carried out the fMRI studies, data analysis and drafted the manuscript. HY and TO conducted the MR imaging. MY, TH and KM prepared the task materials. YY and HI participated in the task design and coordination. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC539237.xml |
314471 | Channelling Evolution | A recent paper suggests that genes can interact in networks to limit variation of phenotype. Similar principles might apply to the regulation of ion channels in nerve cells | Individuals within a wild population show remarkably little morphological variation, given the amount of environmental variation they encounter during development and the amount of genetic variation within the population. This phenotypic constancy led to the proposal that individuals were somehow buffered, or canalized, against genetic and environmental variation ( Waddington 1942 ). Clearly, such a mechanism would have important evolutionary consequences; because natural selection acts upon phenotypic variation within a population, canalization first appears to reduce the evolvability of the trait upon which it is acting ( Gibson and Wagner 2000 ). However, canalization also reduces the effects of new mutations (which may be deleterious), potentially allowing individuals to store this genetic variation without suffering the consequences. If canalization breaks down due to genetic or environmental circumstances, then the stored genetic variation will be released, providing an additional substrate for natural selection. In this way, individuals could potentially undergo large, rapid phenotypic changes. Experiments in both Drosophila and Arabidopsis have suggested that Hsp90 (heat shock protein 90), a member of a family of proteins expressed at high temperatures (heat shock), may be an excellent candidate for bringing about canalization ( Rutherford and Lindquist 1998 ; Queitsch et al. 2002 ). Several features of Hsp90 suggest that it is an evolutionary buffer, capable of hiding and then releasing genetic variation: (1) individuals heterozygous for mutations in Hsp83 (the gene encoding Hsp90) show increased levels of morphological abnormalities; (2) individuals treated with a pharmacological inhibitor of Hsp90 show severe morphological abnormalities; (3) the normal function of Hsp90 is to stabilise the tertiary structure of signal transduction molecules involved in developmental pathways; and (4) this function may be compromised by environmental factors, e.g., heat shock. Gene Networks Generate Canalization Hsp90 may not, however, be uniquely placed to act as an evolutionary buffer producing canalization. Recent theoretical work has suggested that canalization may be an emergent property of complex gene networks and may not require specific mechanisms of protein stabilisation and environmental coupling such as those provided by Hsp90 ( Siegal and Bergman 2002 ). Siegal and Bergman (2002) proposed that when a network is compromised by ‘knocking out’ one of several genes, buffering may be lost or compromised, releasing variation that was hidden in the intact network. To test this, Bergman and Siegal (2003) used numerical simulations of a complex network of ten genes in which each gene is capable of influencing the expression of other genes as well as itself ( Figure 1 A). This network essentially defines the genotype of the individuals within the population, and the amount of gene expression at equilibrium defines the phenotype. Comparison of populations founded by either wild-type individuals or those with a single gene ‘knockout’ revealed much higher levels of phenotypic variation in populations derived from the ‘knockouts’. Figure 1 Similarity between a Gene Network Acting as an Evolutionary Buffer and a Gene Network Regulating Neuronal Ion Channel Expression (A) Each gene (horizontal arrow) is regulated by the products of the other genes by means of upstream enhancer elements (boxes). The strength and direction of regulation (depicted as different colour saturation levels) are a function of both the upstream element and the abundance of its corresponding gene product. (B) A similar representation of a putative network for activity-dependent ion channel regulation in a neuron in which Ca 2+ concentration acts as a feedback mechanism. (C) The mechanism of ion channel compensation in a neuron. The activity of the neuron is dependent upon its synaptic inputs and the suite of ion channels it expresses. Mutation of a gene encoding an ion channel leads to a change in the properties of that channel (depicted as a change in colour saturation) and hence to an increase in activity and internal Ca 2+ (purple). These changes induce a compensatory increase in the expression of another ion channel (red) to restore the original level of activity. Thus, populations derived from ‘knockouts’ express phenotypic variation that was not expressed by the wild-type network, suggesting that any of the genes within the network may buffer genetic variation. This suggests that at least one aspect of generating evolutionary buffering is not unique to Hsp90. But can genes that, unlike Hsp90, are not conditional upon the environment act as evolutionary buffers? To test this, Bergman and Siegal (2003) simulated a gene network that incorporated a mutation process in which single genes may be ‘knocked out’ and then, at a later time, restored. The simulated populations were allowed to evolve whilst being selected for an optimum phenotype (i.e., the populations were exposed to an environment in which a particular phenotype was optimal). A new optimum phenotype was then specified in which the expression of three of the ten network genes changed from on to off or vice versa (i.e., there was a shift in the environmental conditions favouring a different phenotype). Populations evolving with the mutation process reached the new optimum before populations without the mutation process. Thus, the ‘knockout’ mutations were clearly beneficial because they sped up adaptation to a new phenotypic optimum by releasing hidden genetic variation, thereby providing a new substrate upon which natural selection may act. Yet these mutations were not coupled to the new environment, suggesting that the release of the hidden genetic variation does not have to be linked to an environmental change in order to be beneficial. The simulations described by Bergman and Siegal (2003) suggest that the key properties of an evolutionary buffer, the ability to store and then release genetic variation in response to environmental or genetic change, are not unique to Hsp90. Indeed, the simulations suggest that evolutionary buffering may be a widespread property of gene networks. They also suggest that the hidden genetic variation does not have to be revealed by an environmental change, but can be produced by a gene ‘knockout’. These results may go some way to explain the original observation by Waddington (1942) of phenotypic constancy, yet many questions remain ( Stearns 2003 ). One of the major outstanding questions must be whether it is possible to verify these results experimentally. Bergman and Siegal (2003) used data from the yeast Sacchromyces cerevisiae , in which each gene may be ‘knocked out’ in turn and the expression of the remaining genes determined, to demonstrate that their simulations also had application to biological gene networks. Using these data, they showed that ‘knockouts’ show greater variability in gene expression than wild-type yeast, suggesting that buffering has been disrupted. Ion Channels as Evolutionary Buffers Given the results of Bergman and Siegal (2003) , it should be possible to find gene networks in which the elimination of single genes reveals variation in gene expression and hence in phenotype. One class of gene network that may conform to the structure outlined by Bergman and Siegal (2003) is that of the gene networks regulating ion channel expression in neurons. Neurons contain an array of voltage-dependent Na + and K + channels as well as numerous Cl − , Ca 2+ , and voltage-independent leak channels. The electrical properties of a single neuron are dependent, though not exclusively, upon the suite of ion channels expressed within that neuron. The properties of a neural network, which generates behaviour, are determined both by the intrinsic expression patterns of ion channels within neurons and the connectivity between neurons. The nervous system develops as an interaction between experience and genetically programmed events. One mechanism by which this interaction is achieved is ion channel compensation ( Turrigiano 1999 ); individual neurons can change their sensitivity to inputs by altering the relative proportion of ion channels, enabling them to maintain stable properties in the face of changing experience ( Turrigiano et al. 1994 ; Brickley et al. 2001 ; Maclean et al. 2003 ; Niven et al. 2003a ) ( Figure 1 B). Many studies of ion channel ‘knockouts’ show relatively little change in overall neuronal activity, although predictions based upon pharmacological blockade of the ion channels suggest there should be a more severe phenotypic change ( Marder and Prinz 2002 ). Subsequent work has shown that the loss of an ion channel may often be compensated by a change in the expression of other ion channels. For example, the neurons upon which I work are Drosophila photoreceptors. In these neurons, loss of the one particular ion channel leads to compensatory changes in other ion channels linked to the activity of the neuron to restore the ability to process visual information ( Niven et al. 2003a , 2003b ). However, these changes do not restore the original phenotype completely, and the compensated photoreceptors still show a reduced ability to process visual information. In many neurons, it appears that the intracellular Ca 2+ concentration acts as an internal sensor of neural activity ( Marder and Prinz 2002 ). Ca 2+ , along with other second messengers, may influence the expression of genes encoding ion channels, allowing their expression to be coupled to neural activity ( Berridge 1998 ) ( Figure 1 B and 1 C). Additionally, activity-independent mechanisms of ion channel compensation have been described in which the expression of one ion channel is linked to the expression of other opposing ion channels within a neuron ( Maclean et al. 2003 ). These two systems of activity-dependent and activity-independent ion channel compensation bear a close resemblance to the gene network simulated by Bergman and Siegal (2003) in which each gene regulates its own expression and that of other network genes. It is possible, therefore, that the networks of genes regulating ion channel expression may act as evolutionary buffers. The relationship between neural activity and the network of ion channel encoding genes may stabilise the neural activity in relation to both the genetic and environmental variation. The stabilisation of neural activity may have consequences for the generation of adaptive behaviour, which is constructed from neural activity. It is possible that ion channels could canalize the evolution of the nervous system by reducing behavioural variation and therefore removing the substrate on which natural selection may act. For example, changes in voltage-dependent Na + channel properties (such as the activation voltage) may be compensated for by regulating the expression of other ion channels. ‘Knockout’ of one of these compensating ion channels may reveal the change in voltage-dependent Na + channel properties, resulting in a shift in the output of the neuron. This hypothesis has several testable predictions. For example, ‘knocking out’ an ion channel should increase the variation in the activity of particular neurons among individuals in a population. This variation in neural activity may produce an effect on the behaviour of the whole organism. Studying canalization in ion channel gene networks may have significant advantages over studying developmental gene networks because it is relatively straightforward to measure the amounts of ion channels expressed in single identified neurons, to alter the expression of individual ion channels, and to relate these alterations to behaviour. I am currently pursuing the impact of ion channel compensation in Drosophila photoreceptors ( Niven et al. 2003a , 2003b , 2003c ). In this system, changes in ion channel expression produce changes in the coding of visual information, which may lead to behavioural differences. The possible role of ion channel compensation in canalizing the evolution of the nervous system may have important implications not just for understanding this system, but also for understanding the contribution of ion channel compensation to the function of the nervous system and its evolution. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC314471.xml |
524514 | A novel approach to treatment of hypertension in diabetic patients – a multicenter, double-blind, randomized study comparing the efficacy of combination therapy of Eprosartan versus Ramipril with low-dose Hydrochlorothiazide and Moxonidine on blood pressure levels in patients with hypertension and associated diabetes mellitus type 2 – rationale and design [ISRCTN55725285] | Hypertension and diabetes mellitus are closely interrelated and coexist in as many as two-thirds of patients with type 2 diabetes. The consequent risk of such an association is an accelerated development of atherosclerotic cardiovascular disease and nephropathy complications. In choosing an antihypertensive agent, effectiveness needs to be accompanied by favourable metabolic, cardioprotective, and nephroprotective properties. Given the multifactorial nature of hypertension, the approach that has gained widespread agreement is treatment with more than one agent. Agents with different mechanisms of action increase antihypertensive efficacy because of synergistic impacts on the cardiovascular system. Combination therapy allows the use of lower doses of each antihypertensive agent which accounts for the excellent tolerability of combination products. The aim of the present study is to quantify the efficacy of combination therapy of Eprosartan 600 mg respectively Ramipril 5 mg with low-dose Hydrochlorothiazide and Moxonidine on blood pressure levels in patients with essential hypertension and associated diabetes mellitus type 2. The use of monotherapy (Eprosartan or Ramipril) followed by addition of low-dose Hydrochlorothiazide as second agent and of Moxonidine as a third agent will be individualized to the severity of hypertension in the particular patient and to his/her degree of response to current treatment. | Background The clinical combination of hypertension and diabetes carries a particular poor prognosis [ 1 - 6 ]. Clinical studies done in individuals with type 2 diabetes and substudies obtained from clinical trials done in the general population have demonstrated that achievement of goal blood pressure (< 130/80 mm Hg) in this patient category is crucial in decreasing the premature morbidity and mortality [ 7 ]. Thus, management of subjects with type 2 diabetes and associated hypertension needs to be early and aggressive, and must use a global approach. Findings from large, international outcomes studies as well as guidelines and recommendation of prestigious international scientific bodies have made available consensus recommendations [ 8 - 13 ]. The challenge clinicians are facing is to tighten blood pressure control to less than 130/80 mmHg and to adjust initiation of therapy to the severity of hypertension in the individual patient. This multicenter study will evaluate the efficacy and tolerability of monotherapy, double- and triple- antihypertensive combination therapies in a large spectrum of hypertension & diabetes patient population, as summarised in Table 1 . Table 1 Large spectrum of hypertension and diabetes patient population selected for the multicenter study that will evaluate the efficacy and tolerability of monotherapy and double and triple-antlhy pertensive combination therapies Goal BP * Threshold Upper limit for all patients regardless BP values for initiation of double-combination of BP values targeted < 130/80 mmHg > 150/90 mmHg ≤ 179/109 mmHg * The Goal BP defines the cut off point for responders / non-responders to any therapy. Table 2 (see Additional file 1 ) specifies the treatment strategies to be employed in the study as adjusted to severity of hypertension in the particular patient and to his/her degree of response to that therapy. The primary objectives of hypertension management in patients with diabetes are to reduce blood pressure levels to currently recommended target level and thus to reduce the risk of cardiovascular and renal complications without adversely impacting glycemic and lipid control. Previous debate regarding the level of blood pressure reduction that optimizes cardiovascular risk reduction is currently settled. BP goal of < 130/85 mmHg promoted by the JNC-VI guidelines issued 1997 [ 10 ] were replaced in 2002 by a position paper of the American Diabetes Association (ADA) supporting a target blood pressure in hypertension & diabetes patients of < 130/80 mmHg [ 14 ]. This blood pressure-goal is also endorsed by the most recent JNC-7 guidelines [ 15 ] and two other American professional societies [ 16 , 17 ] as well as by the ESH/ESC [ 9 ] and formally by the ISH. A widespread agreement, supported by the above mentioned organizations/societies is in place, regarding the principles governing the use of appropriate antihypertensive drug combinations to maximize hypotensive efficacy while minimizing side effects. Polypharmacy is common place and, with at least one third of patients requiring two or more agents simultaneously, a paradigm shift in the approach of initiating therapy is done by advocating use of two agents in subjects with more severe hypertension (BP in excess of 20/10 mmHg above goal). Low-dose thiazide diuretic is favored as one of the two starting agents. In general, monotherapy is likely to be successful in mild hypertensive patients (grade 1 hypertension) without associated major risk factors for CHD. In contrast, patients with type 2 diabetes need more rigorous control of BP in an easier, simpler fashion, given the remarkable complexity of the multiple drug regimens needed to control their comorbid medical problems (e.g., diabetes, obesity, high cholesterol). A large body of evidence derived from a multitude of international trials have demonstrated both the benefit of low-level, goal blood pressure, in terms of prevention of long-term complications and, the need for multiple drug combinations in order to achieve that goal [ 13 , 18 - 20 ]. Furthermore, in a computer-modelled cost-effectiveness analysis of the JNC-VI treatment goal (< 130/85 mmHg), lowering blood pressure to goal increases patients' life expectancy and decreases long-term cost [ 21 ]. Cost-effectiveness analysis in the context of the UKPDS study has also revealed that incremental cost of tight control (< 150.85 mmHg) versus less tight control (< 180/105 mmHg) was considered to be effective [ 22 ]. In the HOT study [ 13 ], which recruited grade 2 and 3 hypertensives after washout from previous agents, monotherapy was successful in only 25–40% of patients, according to the target diastolic blood pressure. In trials of diabetic patients, the vast majority were on at least two drugs, and, in two recent trials on diabetic nephropathy [ 23 , 24 ] an average of 2.5 to 3.0 non-study drugs were required in addition to the angiotensin receptor antagonist used in these studies (losartan/irbesartan). Given the very poor BP control rate, i.e., 11% in patients with hypertension & diabetes, the use of combination therapy is an important therapeutic consideration, as it facilitates quicker and easier attainment of goal BP and should lead to a greater proportion of people with diabetes who achieve BP goal. Initiation of treatment by combination therapy was effectively tested in the VA study at the beginning of the antihypertensive treatment trial era [ 25 , 26 ] and recently in the PROGRESS study [ 27 ]. Methods Patient Population Subjects will be recruited in outpatient clinics/offices of general practitioners and internal medicine/cardiology specialists from the entire spectrum of patients having coexistent hypertension and diabetes mellitus type 2. The upper limit of blood pressure values targeted (≤ 179/109 mmHg) correspond grade 2 hypertension (according to current ESC/ISH and WHO guidelines). Subjects to be recruited are supposed to have both entities (hypertension with BP values in the range ≥ 130/80 – ≤ 179/109 mmHg and diabetes mellitus type 2) diagnosed since previously, undergoing current antihypertensive treatment and, to be eligible for the study according to the specific inclusion/exclusion criteria described below. The study consists of five distinct phases: Screening (S) (up to seven days), Placebo Run-in phase (two weeks), monotherapy (four weeks, double-blind fashion), double-combination treatment (Eprosartan/HCTZ respectively Ramipril/HCTZ for four weeks, HCTZ open labelled), triple-combination treatment (Eprosartan/HCTZ/Moxonidine versus Ramipril/HCTZ/Moxonidine with HCTZ and Moxonidine open labelled) and Follow-up (two to seven days). Patients allocated to monotherapy will participate in the study for a four weeks period while those starting with double combination therapy will receive medication for a maximum of 12 weeks. The flowchart below captures the main events during the study conduct (Fig. 1 ). Figure 1 Study Design. A multicenter, double-blind, randomized study comparing the efficacy of combination therapy of Eprosartan versus Ramipril with low-dose Hydrochlorothiazide and Moxonidine on blood pressure levels in patients with essential hypertension and associated diabetes mellitus type 2. Randomization and Blinding Randomization will be concealed. A stratified randomization will be employed based on the following rules: 1. Subjects with blood pressure in range: BP ≥ 130/80 – ≤ 150/90 mm Hg will be randomly allocated to one of the monotherapy arms (Eprosartan or Ramipril). 30 subjects will be recruited for each arm. 2. Subjects with blood pressure in the range: BP > 150/90 – ≤ 179/109 mm Hg will be randomly allocated to double-combination therapy (Eprosartan/HCTZ or Ramipril/HCTZ); 190 subjects will be recruited for each arm. The randomization lists will be provided by the Department of Clinical Supplies at Solvay Pharmaceuticals BV with the program ADLS. Patients will be allocated in equal numbers to each sequence. A fixed block size of patients will be used, and only complete blocks of study medication will be provided to the centers. Within each center, randomization numbers will be used in ascending order and patients will be allocated to randomization code numbers in chronological order. The study will be unblinded when all CRFs are in house and the data on the database have been declared clean. The following drugs are to be used in the study: • Eprosartan 600 mg, once daily • Ramipril 5 mg, once daily • Hydrochlorothiazide 12.5 mg, once daily • Moxonidine 0.4 mg, once daily • Placebo tablets matching Eprosartan 600 mg and Ramipril 5 mg will be used in the monotherapy, in the double- and in the triple-combination therapy phases. Hydrochlorothiazide tablets will be open labelled in the double- and triple-combination therapy phases. Moxonidine tablets will open-labelled in the triple-combination therapy phase. Eprosartan and Ramipril and the corresponding placebos will be packaged according to the double-dummy technique. Fig. 2 summarizes the treatment algorithm. Figure 2 Treatment Algorithm Monotherapy (Eprosartan vs. Ramipril) Patients eligible for participation in the study by the assessment at V2 will be randomized on the basis of the severity of their hypertension and allocated to either monotherapy or to double-combination therapy (described below). Patients with initial blood pressure values in the range ≥ 130/80 – ≤ 150/90 mmHg, will be randomly allocated to one of the monotherapy groups. By the end of the first four-week phase an assessment will be made as to whether patients have reached or not the "goal" hypertension (< 130/80 mmHg). Patients on monotherapy deemed to be responders at the end of four weeks treatment (i.e., have reached the "goal") will terminate their participation in the study and be followed up during the ensuing two to seven days after stopped monotherapy (procedure similar with Follow-up at end of study (V6). Non-responders (according to above mentioned criteria, i. e., with BP still ≥ 130/80 mmHg), will receive double-combination therapy (Eprosartan/HCTZ respectively Ramipril/HCTZ) and will be followed-up for eight weeks (red-dotted line in the flowchart). Double-combination Therapy (Eprosartan/HCTZ vs. Ramipril/HCTZ) Patients with initial blood pressure values in the range > 150/90 – ≤ 179/109 mmHg, will be randomly allocated to double-combination therapy. By the end of this first four-week phase an assessment will be made as to whether patients have reached or not the "goal" hypertension (< 130/80 mmHg). Responders will maintain double-combination therapy and will be followed-up for an eight week period (V3 toV5, dotted-line in the flowchart) and retain therapy unchanged. Non-responders will receive triple-combination, as described below. Triple-combination Therapy (Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine) Non-responder patients after four weeks of double-combination therapy will receive triple-combination (Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine) and will be followed-up for an eight weeks period (V3 to V5). After the first four weeks of monotherapy respectively double-combination therapy (V3), patients will be reassessed for compliance, adverse events and supplied with medication for the next eight weeks (except for the monotherapy patients who reached goal blood pressure and who will terminate the study). During the eight weeks triple-combination therapy all patients will be reassessed for compliance and adverse events (visits V4, V5 and V6). A 12-lead ECG will be performed by S, V3 and V5 while safety laboratory parameters will be performed by S, V2 and finally by visit V5. A Follow-up Visit will be performed on all patients with full physical examination, BP and pulse rate check within the ensuing two to seven days after study end (V6). Further follow-up and optimal treatment will be decided on a case-by-case basis by the physician in charge. Table 3 (see Additional file 2 ) displays a summary of the scheduled investigations, as planned for each particular visit. Inclusion Criteria 1. Males and females aged 40 to 80 years of age. Women of childbearing age will be subject to pregnancy testing and will agree to maintain adequate hormonal contraception. 2. Eligible patients should have diagnosed essential hypertension (not controlled with current treatment, i.e., BP ≥ 130/80 – ≤ 179/109 mmHg) and diagnosed associated diabetes mellitus type 2, willing to accept withdrawal of any antihypertensive medication by the time of the Screening visit. Exclusion Criteria A multitude of exclusion criteria, carefully listed in the study protocol, can be summarised in three different groups: 1. Ineligibility based on hypertension grade 3 (BP ≥ 180/110 mmHg), any form of secondary hypertension or hypotension (SBP ≤ 90 mmHg). 2. Any form of organic heart disease requiring medical treatment that might have hypotensive effect, imply need for invasive investigation or surgery. 3. The patient is suffering from a severe concomitant illness related to any body organ or system, likely to affect outcome assessment. Likewise, ineligibility is declared for patient anticipated to have compliance problems, participants in another trial during the past 30 days, pregnancy and lactations and known hypersensitivity to ingredients of any of the employed agents (eprosartan, ramipril, hydrochlorothiazide, moxonidine). In addition, diabetes mellitus type 1 is exclusion criteria. Study Outcomes Prior and Concomitant Therapy The study protocol calls for every patient to be treated optimally by the physician in charge and to receive comprehensive, individualized lifestyle change advice regarding relevant diet and physical activity. Visits are to be scheduled in the context of the study (at four weeks interval during ongoing treatment). Any antihypertensive medication should be withdrawn latest by Screening visit and will be prohibited during the whole period of ongoing study. Ethics and Informed Consent The study will be conducted in accordance with ICH GCP and the European Directive 2001/20/EC of the European Parliament and of the Council of 4 April 2001 (on the approximation of the laws, regulations and administrative provisions of the member states relating to implementation of good clinical practice in the conduct of clinical trials of medicinal products for human use) and on the basis of ethical principles laid down in the current revision of the Declaration of Helsinki (Edinburgh 2000). In addition, Solvay Pharmaceuticals GmbH policies and procedures should also be followed. Written consent, involving provision of detailed information regarding the study objectives, design, scope of the intervention, risks and benefits, will be obtained for all patients before initiating any study procedures. Likewise, study documentation is to be subject to the scrutiny of local ethical committees in the two countries participating in the study. Sample Size and Statistical Analysis Efficacy The primary objective is to demonstrate the superiority of combination therapy of Eprosartan/HCTZ (600/12.5 mg) versus Ramipril/HCTZ (5/12.5 mg) with the primary parameter of attention being the percentage of patients brought to goal blood pressure (<130/80 mmHg) at visit V3. Null hypothesis: H 0 : P E+HCTZ = P R+HCTZ Alternative hypothesis: H 1 : P E+HCTZ ≠ P R+HCTZ , Where P E+HCTZ is the percentage of patients brought to goal blood pressure at visit 3 with Eprosartan/HCTZ and P R+HCTZ is the percentage of patients brought to goal blood pressure at visit V3 with Ramipril/HCTZ. The primary parameter will be analyzed using the Cochran-Mantel-Haenszel test, controlling for center effects. Statistical significance will be assessed with a two-sided test at 0.05 α level. The confirmative analysis of the primary parameter will be performed on the intent-to-treat patient sample. Secondary efficacy objectives: • To compare the mean change in sitting systolic blood pressure (sitSBP) and sitting diastolic blood pressure (sitDBP) between Eprosartan/HCTZ and Ramipril/HCTZ (V3 vs. V2). • To compare the percentage of patients brought to goal blood pressure by the triple-combination Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine (V5 vs. V3). • To compare the mean change in sitSBP and sitDBP between triple-combination with Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine (V5 vs. V3). • To compare the mean change in sitSBP and sitDBP between monotherapy with Eprosartan vs. Ramipril (V3 vs. V2) • To compare the percentage of patients brought to goal blood pressure at visit V5 between Eprosartan/HCTZ and Ramipril/HCTZ in patients not at goal blood pressure after four weeks of monotherapy. • To compare the mean change in sitSBP and sitDBP as well as the responder rate in patients non-responders (not at goal ) after four weeks of monotherapy (switched to double combination therapy) (V5 vs.V3); and to compare the mean change in sitSBP and sitDBP as well as the responder rate maintenance in patients who reached goal blood pressure value at the end of first four weeks of double-combination therapy and successively entered an eight weeks follow-up period (V5 vs.V3). Changes in blood pressure parameters will be assessed by analysis of covariance (ANCOVA). The model will include the intercept, treatment and center as fixed effects and the baseline value as covariate. For response rates, the treatment groups will be compared using the Cochran-Mantel-Haenszel test, controlling for center effects. Comparisons of the medication regimens will be reported along with 95% confidence intervals of the relative risk ratios. These analyses will be considered as exploratory. Safety All patients who receive at least one dose of double-blind medication will be assessed for clinical safety and tolerability. Evaluation of safety data will be based on comparisons of patient experience by treatment group. Clinical interpretation of safety will be based on reviews of standard displays of adverse events incidence, pulse rate data, and laboratory test values. Summary statistics of laboratory test values and incidence of adverse events according to treatment and time of onset will be presented. Sample Size, Power and Level of Significance A formal sample size estimation has been done for patients with blood pressure in the range: > 150/90 mmHg and ≤ 179/109 mmHg. Assuming that 55% of the patients in the Eprosartan/HCTZ group would reach goal blood pressure as compared with only 40% in the Ramipril/HCTZ group, a 0.05% two-sided significance level with 80% power to detect the targeted 15% difference will imply the need for 346 patients supposed to complete the four-weeks double-combination therapy phase. Further 35 patients (10% of the total) will be recruited to account for drop-outs. In addition, 60 subjects with BP ≥ 130/80 and ≤ 150/90 mmHg will be randomly allocated to either Eprosartan or Ramipril monotherapy group at visit V2. Inclusion of monotherapy phase with a relatively low number of patients (30 subjects per arm) is justified by the intention to therapeutically target the whole spectrum of patient population having coexistent diabetes mellitus type 2 and mild to moderate hypertension, in whom the agents tested are likely to be effective. Blood Pressure Measurements Office blood pressure will be determined by Riva-Rocci method with a mercury or a mercury calibrated sphygmomanometer throughout the study. All measurements will be made on the same arm supported at heart level, using the same cuff size and the same equipment. If the patient's arm circumference is > 32 cm, a large blood pressure cuff should be used. Diastolic blood pressure will be measured at the disappearance of Korotkoff sounds phaseV. Measurements should be taken by the same staff member at the particular visits. For an individual patient blood pressure measurements should be performed at 24 hours after the last oral dose, at the same time (± 2 hour) in the morning, between 8 and 10 am. Blood pressure will be measured in the following sequence: after the patient sits quietly for at least 5 minutes, blood pressure will be measured twice at approximately 2-minutes interval. The average of these measurements will be recorded. If the difference between measurements is in excess of 5 mmHg a third reading will be performed and the average value recorded as mean sitting systolic and diastolic blood pressure. Measurements should be performed by the same study assistant using the same device, in each of the centres involved in the study. Discussion The current evidence base is strongly in favour of combining drugs in order to achieve blood pressure goals, in particular in patients with coexistent hypertension & diabetes. Likewise, there is a widespread agreement in the scientific community as to the goal blood pressure to be achieved in these patients. Further, common sense in clinical practice dictates that combination therapies should be tailored to severity of hypertension in the individual patient and that, eventual associated risk factors/comorbidities should be accounted for in the process of treatment decision making. Patients with high blood pressure and associated impaired glucose tolerance or overt diabetes mellitus type 2, as a group, are insulin resistant, [ 28 ] glucose intolerant [ 29 - 31 ], hyperinsulinemic [ 32 - 36 ], dyslipidemic [ 37 - 42 ] and with evidence of endothelial dysfunction [ 43 , 44 ]. Extensive epidemiological evidence indicates that diabetic individuals with hypertension have greatly increased risk of cardiovascular disease, renal insufficiency, and diabetic retinopathy [ 45 - 47 ]. For every 5 to 10 mmHg decrease in systolic blood pressure achieved with diuretics, ARBs, ACE inhibitors, beta-blockers or calcium channel blockers in patients with diabetes, there is a 20% to 30% relative risk reduction in cardiovascular events [ 48 - 53 ]. Agents belonging to the nine, most well-known different antihypertensive drug classes produce a similar reduction in systolic and diastolic blood pressure (10–15 and 5–10 mm Hg respectively). Differences in terms of magnitude of blood pressure lowering, as indicated by results from comparative efficacy studies, are usually small [ 54 ]. However, larger differences have been shown as to effects on hard endpoints (myocardial infarction, heart failure, stroke). Comparisons between different agents in patients with hypertension & diabetes mellitus type 2, convincingly point to ACE inhibitors and ARBs as being the two classes of antihypertensive drugs that reduce the activity of the renin-angiotensin II system, and should be among the preferred first-step drugs for the treatment of these conditions [ 55 ]. Angiotensin II increases blood pressure by enhancing aldosterone synthesis, resulting in sodium retention and direct vasoconstriction. The first step in this pathway is inhibited by adrenergic blockers. The third and forth steps are inhibited by ACE inhibitors and ARBs, respectively [ 56 ]. Clinical trials carried out world-wide have shown that ACE inhibitors have renoprotective effects [ 57 , 58 ] and clear cardiovascular benefits [ 59 - 63 ]. Their main side effects are dry cough and angioedema. In contrast, in placebo-controlled trials, the ARBs have demonstrated almost no side effects [ 64 ]. Both ACE inhibitors and ARBs have been shown to maintain quality of life of hypertensive patients equal to or better than other classes of antihypertensive drugs [ 65 - 68 ]. The only laboratory abnormality that may occur with agents from both classes is mild hyperkaliemia, especially in some elderly patients with type 2 diabetes who have hyporeninemic-hypoaldosteronism [ 69 ]. Use of low-dose thiazide diuretic (< 25 mg) as a second agent in treatment of patients with hypertension & diabetes is well-documented and widely recommended [ 21 , 70 - 73 ]. It has beneficial effects on both morbidity and mortality figures while, previous general concern on the negative impact of diuretics on the different lipid parameters is no longer justified as, all long-term studies with low-dose diuretics have not been shown to affect lipid profiles in a negative way [ 74 - 76 ]. Moreover, in studies of a year or more, diuretics have been shown to reduce cardiovascular risk in every trial to date [ 77 - 79 ]. Since drug combinations may be required for many years in the age-groups in which type 2 diabetes is most prevalent, there have been calls for the use of agents devoid of adverse effects on carbohydrate and lipid metabolism. It has been suggested that such effects may account for the shortfall in reduction of coronary heart disease observed in clinical trials of diuretics and β-blockers [ 80 , 81 ]. Moxonidine stimulates imidazoline-I 1 receptors in the medulla, thereby reducing central sympathetic drive and attenuating peripheral vascular resistance. In addition, reduced sympathetic drive results in lower plasma concentrations of catecholamines and renin. Randomised comparative studies show that the efficacy of moxonidine as monotherapy is similar to that of other antihypertensive agents [ 82 ]. Moreover, selectivity for the I 1 receptor greatly reduces the adverse affects attributable to costimulation of medullary α 2 -adrenoceptors [ 82 ] observed with the first generation of centrally acting agents, α-methyldopa and clonidine. In clinical studies, moxonidine has been shown to have neutral or beneficial effects on lipid and carbohydrate metabolism [ 82 , 83 ]. A retrospective analysis suggested minor dose-dependent reductions in fasting plasma glucose in moxonidine-treated hypertensive patients. On the available evidence, moxonidine seems to be a logical choice as component of combination treatment of patients with hypertension and associated diabetes mellitus type 2 or impaired glucose tolerance. Conclusions The poor blood pressure control in patients with hypertension & diabetes in everyday life lies, at least in part, in the emphasis in the evidence-based guidelines of the recent past towards advice on initial, single treatments as well as in their lack of clarity and transparency in recommending pre-specified blood pressure targets [ 10 , 84 - 86 ]. Previous consensual advice that combination treatments expose patients to the increased risk of adverse events has been replaced by good evidence to the contrary: use of several agents combined or of fixed-dose combinations treatments have the potential to bring patients to goal blood pressure and thereby to minimize long term risk of hypertension/diabetes-related complications [ 22 - 27 , 87 ]. Despite the apparent simplicity of the paradigm shift towards clear blood pressure goal and individualized therapy on the basis of hypertension severity (and addition of a third agent in case of uncontrolled BP with two agents), comparative data to guide clinical practice is still lacking and this applies also to the comparison of ARBs versus ACE inhibitors. The present study attempts to explore this area. Competing interests Pater C, Berrou JP, Luszick J and Beckman K are employees of Solvay Pharmaceuticals. Authors' contributions Study concept and design: Pater, Berrou, Luszick Drafting of manuscript: Pater, Bhatnagar Statistical expertise: Beckman Supplementary Material Additional File 1 Table 2 – Blood pressure-adjusted treatment stratification Click here for file Additional File 2 Table 3 – Investigations Schedule Click here for file | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524514.xml |
549539 | Comparison of knowledge scores of medical students in problem-based learning and traditional curriculum on public health topics | Background The purpose of the study was to compare the knowledge scores of medical students in Problem-based Learning and traditional curriculum on public health topics. Methods We planned a cross-sectional study including the fifth and sixth year medical students of Dokuz Eylul University in Turkey. The fifth year students (PBL group, n = 56) were the pioneers educated with PBL curriculum since the 1997–1998 academic year. The sixth year students (traditional education group, n = 78) were the last students educated with traditional education methods. We prepared 25 multiple-choice questions in order to assess knowledge scores of students on selected subjects of Public Health. Our data were collected in year 2002. Results Mean test scores achieved in PBL and traditional groups were 65.0 and 60.5 respectively. PBL students were significantly more successful in the knowledge test (p = 0.01). The knowledge scores of two topics were statistically higher among PBL students. These topics were health management and chronic diseases. Conclusion We found that mean total evaluation score in the PBL group was 4.5 points higher than in the traditional group in our study. Focusing only on the knowledge scores of students is the main limitation of our study. Upon the graduation of the first PBL students in the 2002–2003 academic year, we are planning additional studies regarding the other functions of a physician such as skill, behaviour and attitude. | Background During the last 25 years, ideas concerning the aim, structure and system of medical education have been discussed. Debates generally have arisen from the perception that medical education couldn't serve the purpose of improving health standards of the communities [ 1 ]. "Health for All" was adopted in 1977 and launched at the Alma Ata Conference in 1978 to underline the fact that large numbers of people and even whole countries were not enjoying an acceptable standard of health [ 2 ]. In order to achieve the goal of "Health for All" and to improve the health standards, medical schools must provide physicians who are familiar with the community and its health problems, their prevention and solutions. Then their curriculum must be expedient to this goal [ 3 , 4 ]. World Health Organization (WHO) also emphasizes the fact that medical students must be educated considering the health needs of the population in which they live [ 5 ]. In the Edinburgh Declaration of the World Medical Association in 1988, similar problems were mentioned and the purpose of the medical education was declared as training physicians capable of improving communities' health standards. This declaration suggested that medical education should be focused on common health problems of the large communities, and the medical school curriculum should be restructured according to the health requirements of the community. According to the declaration, medical students must gain professional skills and social values in addition to theoretical knowledge and the principle of lifelong medical education should be adopted [ 6 ]. The ideas and suggestions mentioned above have aroused strong winds of change in the medical education arena. Mc Donald et al. from Mc Master University determined an approach based on the community's main health problems and stressed the importance of focusing on these problems while designing their medical school's curriculum [ 7 ]. Since then, this approach has been adopted by many medical schools all over the world. The schools which designed their curriculum according to the priority health problems of the community, managed to raise the physicians' awareness of their community and the preventive measures and solutions of their main health problems. In Turkey, problems of medical education have been discussed since early 1970s. Several studies showed that the goals of medical education did not overlap with the health requirements of the Turkish community. The education of health professionals was abstracted from the realities of the country. In 1990s Turkish Parliament and Turkish Medical Association determined and reported the difficulties of medical education. In a 1991 report of the Turkish Parliament, the facts that the number of qualified physicians who were trained according to the health needs of the country was limited and that this number was not sufficient to improve its health standards were underlined. Several deans from different medical schools of the country contributed to Turkish Parliament's study and reported that a greater importance should be given to the health problems of the population while planning the educational programs and the medical education should not be restricted to the university hospitals [ 8 ]. In The Turkish Medical Association's report the fact that medical education was not relevant to health needs of the country was emphasized. New medical graduates were not fully aware of common national health problems. The recommendations of the Turkish Medical Association to improve the health standards of the Turkish population were; training the general practitioners capable of working effectively in the primary health care and restructuring the medical education on a community basis and implementing Problem-based Learning methods [ 9 ]. International developments and the reports of Turkish Parliament and Turkish Medical Association led the faculty of Dokuz Eylül University School of Medicine (DEUSM) to seek solutions to the problems mentioned in the reports. As a result, Problem-based Learning (PBL) a more active and student-centred learning- was adopted and launched in the 1997–98 academic year. One of the main features of the education program was its relevancy to the philosophy of community-based medical education [ 10 ]. The curriculum of DEUSM was structured considering social, biological, behavioural and ethics objectives of medical education. The curriculum was structured in a modular system and adopted to a spiral configuration providing horizontal and vertical integration. During the first three years of undergraduate education, PBL sessions are the main focus of a modular structure. The weekly schedule of a module allowed for all the educational activities such as PBL sessions, lectures, field studies, communication skills and clinical skills courses lectures existing one hour a day in the weekly program support the PBL sessions and independent learning [ 11 ]. PBL sessions were based on written problems, which are likely to happen in real life. Special emphasis was also given to the integration of knowledge, acquisition of professional and moral values and to the development of communication skills. Medical knowledge and practical skills that a physician is supposed to have were on the basis of the advice of Turkish Medical Association and the faculty departments. The Department of Public Health also contributed to the education program by setting social standards and determining the most important health problems of the community. PBL Curriculum of DEUSM aimed to teach the students the main health problems of the community, their prevention and ways of treatment. Public Health topics of Dokuz Eylül University School of Medicine consists of; • Holistic approach in health, • Basic principles of Public Health, • Personal and social points of view on health events, • Bio-psychosocial (holistic) approach to any individual, • Principles of preventive medicine, • Structure and mechanisms of national health organization, • Demographic structure and trends, factors affecting them, • Basic principles of planning and conducting a scientific research on health, • Sound knowledge on leading health problems of the country, personal and social approaches for their solutions, • Environmental and occupational factors threatening community health and their prevention. Cases in the scenarios of the PBL modules were selected among common and important health problems, for which early diagnosis or prevention is possible. Lectures and small group studies with students were also organized to contribute to the educational effectiveness of the modules. Public Health topics of the medical education may be achieved more easily when theoretical knowledge and practical skills are complemented by field studies [ 12 ]. It is recommended to start such activities as early as possible and to continue them during medical education. In DEUSM Public Health perspective, objectives of each academic year were determined and relevant field study programs were developed to contribute these objectives. These programs were put into practice beginning from the beginning of the medical education. Prior to the implementation of PBL curriculum in the 1997–1998 academic year, lectures on Public Health were presented to the first, the third and the last year students by the faculty members of the Department of Public Health. Lectures on bio-statistics and research methods were given weekly throughout the first year. The other topics of Public Health were held in 72-hour Public Health Courses at the end of the third year [ 13 ]. In the new curriculum public health subjects were held in PBL sessions. Each PBL scenario had at least one chapter associated with public health issues. Another difference between traditional and problem based education methods was changing roles of the students and teaching. Traditional education was teacher based and the students were passive receivers while the lecturer was giving information. But in PBL method, roles were exchanged and the sessions were carried out by noninformative teachers and more active students. Comparison of old and new curriculum using some measurement tools is mandatory to observe the effects of innovations. In the literature, the determination of students' performances in scientific or licensing examinations was used to compare the efficiency of traditional education and PBL. Nandi P. et al. reviewed the studies and meta-analyses comparing PBL and traditional lecture-based education methods. In meta-analysis of the data published between 1980–1999, they concluded that PBL helped students show slightly but not significantly better performance than the others on clinical examinations [ 14 ]. Similar results were reported by Albanese M. et al., in a meta-analytic study evaluating published data between 1972–1992 [ 15 ]. Blake et al. compared formerly lecture-based educated and recently Problem-based educated graduates of Missouri-Columbia School of Medicine concerning their performances on medical licensing examinations. They reported that mean scores achieved on these examinations were better among graduates of PBL, but the difference between old and new graduates' scores was not statistically significant [ 16 ]. Some other studies have attempted to compare students' performances on special areas of medicine instead of general evaluation. Antepohl and Herzig conducted a randomized controlled study among the students who enrolled for the course of basic pharmacology at the University of Cologne. They randomly divided the students into two groups of PBL and traditional lecture based learning in order to compare their final examination scores. They could not find any significant difference between the two groups. However, in short essay questions there was a tendency towards higher scores among the students in the PBL group. The authors also found that the PBL students reached almost identical scores in their multiple choice questions and their short essay questions whereas the students who had been in the lecture based group scored significantly lower scores in their short essays than in their multiple choice questions [ 17 ]. In a multi-centric study conducted by Schmidt et al., comparison of PBL and lecture based learning students showed that PBL students had higher knowledge scores on the areas of primary care services, psychological health, collaboration of different sectors on health and occupational ethics [ 18 ]. The purpose of our study was to compare the knowledge scores of medical students in PBL and traditional curriculum on public health topics. Methods We planned a cross-sectional study including the fifth and sixth year medical students of DEUSM. The fifth year students (PBL students) were the pioneers educated with PBL curriculum since the 1997–1998 academic year. The sixth year students (traditional education group) were the last students educated with traditional education methods. The knowledge scores of students on Public Health topics were evaluated. In both of the PBL and Traditional curriculum, all the knowledge acquired in the first five years of the school was reviewed during the two-month Public Health internship period in the sixth year. Since this period may remind the students of some issues which may have been previously forgotten, we decided to exclude the sixth year students who have completed their internship period. 56 fifth year students and 78 sixth year students who have not so far completed their internship period in the Department of Public Health were included in our study. Participation rates were 96.4% (54 out of 56 students) in the fifth year and 100% (all of the students) in the sixth. Before the application of the inquiry form, the purpose of the study was explained to the students and their oral consents were obtained. We analyzed the knowledge scores of the two groups of students' on Public Health issues. PBL and traditional programs were the independent variables. Descriptive variables were age and gender. By reviewing a five yearlong section of educational programs, we determined that nine Public Health main topics were common to both PBL and traditional programs. The main topics were communicable diseases, epidemiology, mother and child health, health management, chronic diseases, occupational health, nutritional principles in community, demography and environmental health. We prepared 25 multiple-choice questions in order to assess knowledge scores of students on selected subjects. The number of questions related to each topic was proportional to the time allocated for each of the topic in the curriculum. The content validity of the questions was tested by consulting experts in relevant fields. All the data were collected between February and March 2002. Scoring procedure was implemented over "100 points" where each correct answer was scored "four points" and each wrong answer was scored "zero point". Data were subjected to statistical analysis by the chi-square test and the t-test in SPSS 10.0. Results Overall mean age was 23.6 ± 2.1 (21–45) years. The rates of male and female students were 55.4 % and 44.6 % respectively. There were no statistically significant differences between the two groups regarding mean ages, gender distribution or other personal variables. Mean scores achieved at the 25 question-test were 65.0 in PBL group and 60.5 in the traditional group. Students in the PBL group were significantly more successful in the knowledge test (Table- 1 ). Table 1 Comparison of mean scores of the students in PBL and traditional programs Topics Max. point for each topic PBL Traditional t p Mean Score (±) SD Mean score (±) SD Communicable diseases 20 12.5 4.32 12.3 3.74 0.290 0.77 Epidemiology 20 6.2 4.23 5.4 4.13 0.990 0.32 Mother and child health 20 14.9 3.89 15.5 3.70 -0.851 0.39 Health management 12 9.6 2.74 7.7 3.34 3.447 0.00 Chronic diseases 8 6.7 2.17 5.3 2.78 3.255 0.00 Occupational health 8 6.1 2.54 5.7 2.46 0.743 0.45 Nutritional principles in community 4 1.9 2.01 2.0 2.01 -0.416 0.67 Demography 4 3.3 1.50 2.9 1.75 1.257 0.21 Environmental health 4 3.6 1.17 3.3 1.45 1.070 0.28 Total 100 65.0 10.99 60.5 9.22 2.395 0.01 The knowledge scores of seven topics were higher among students in PBL curriculum. These topics were communicable diseases, epidemiology, health management, chronic diseases, occupational health, demography and environmental health. Traditional curriculum students were found to be more knowledgeable on two topics; mother and child health and nutritional principles in the community. However, the differences between PBL and traditional students' knowledge scores in only two topics, chronic diseases and health management, were statistically significant (Table- 1 ). Conclusions In our study, we found a statistically significant difference between knowledge scores of PBL and Traditional education groups in favour of the PBL group (Table 1 ). The students of the PBL group had higher knowledge scores on 7 of the 9 identified topics. But the difference between mean scores of the groups was statistically significant in only two topics, "health management" and "chronic diseases". The reason of significantly higher knowledge scores among the students in PBL group may be that these students have more opportunities such as observations during field studies, work-shops or presentations to study on these two topics than those in the other group. They experienced a two week training period in a "community health center" at the end of the first year and observed the health center services and prepared a structured form concerning the procedures of health centers. They also studied in "community health centers" as small groups including two students in each fortnightly during their third year in the school and completed comprehensive forms about the topics on which they studied. The reason of better knowledge scores of PBL group on "chronic diseases" may result from the special educational efforts improving the effects of relevant modules on this topic. Actually special learning opportunities were provided for all topics and we were expecting to find a difference on remaining 7 topics too. On the other hand, the students in the traditional education group had slightly higher mean scores about the topics of "mother and child health" and "nutritional principles in community" although the differences between the groups' mean scores were not statistically significant. These knowledge deficiencies among PBL students were already revealed and an additional module was implemented in the curriculum to compensate them. Curriculum of DEUSM is being looked over by curriculum committee continuously and the departments try to make interventions for problematic parts. We found that the mean total evaluation score in the PBL group was 4.5 points higher than in the traditional group in our study. Actually, we expected a much larger difference between the two groups in favour of PBL students for their education was supported by lectures, small group studies and field studies in addition to the PBL sessions. They also had the advantage of studying on Public Health issues in each year of the school by means of homogenous allocation of the modules and blocks in the first five years instead of accumulation in a short period of time as it was in the traditional curriculum. Therefore, the difference between the evaluation scores of the groups did not meet our expectations although it was statistically significant. The reason for this underachievement of Public Health objectives among our PBL students may be related to both students and PBL tutors. The common perception among the students that they have enough knowledge to say something about social and behavioural aspects of PBL modules lead them to focus on biological objectives more and they do not need to study on social issues in depth. Furthermore, a common misunderstanding among faculty members that achieving the Public Health objectives in PBL is just the responsibility of the Department of Public Health may have led the PBL tutors to withdraw from the responsibility of focusing on these subjects sufficiently. Additionally, when they are less informed or less equipped with supporting material about Public Health objectives, they may not have felt very competent while facilitating their groups by asking appropriate questions. One assumption of curricular comparison studies, included this one, is that students will do better either in one or the other type of curriculum. However, each curriculum demands different skills and deployment of learning strategies from the students. This is important because, it is well known in the educational literature that not all students do well in one particular learning program and that they do better when the program adapts to their preferred way of learning. The studies of learning styles may shed light in why the differences between performance scores are always so close when medical curricula are compared. As we mentioned before, in DEUSM, the written problem used in PBL sessions are oriented to biological as well as social and behavioural objectives. In order to achieve all these three objectives the tutors must attach the same importance to each subject and ensure that their groups give enough time and effort for each objective. But when the tutors get inadequate information and support from the experts of the related subjects, they generally focus only on biological objectives and their groups can't manage to integrate all objectives. If the tutors are less sensitive to objectives other than biological ones, then their students will be less motivated to learn and, like their educators, will be equally insensitive to Public Health topics. In order to prevent this, faculty members of the department of Public Health who take place in the scenario committees review the PBL problems regarding Public Health objectives. They make every effort to insure that the Public Health objectives are included while writing the problems and that the tutors are sufficiently informed on these objectives before their sessions. Field Work Committee has been trying to increase students' motivation and raise their awareness on Public Health issues to increase the effectiveness of field studies. Focusing only on the knowledge scores of students is the main limitation of our study. Upon the graduation of the first PBL students in the 2002–2003 academic year, we are planning additional studies regarding the other functions of a physician such as skill, behavior and attitude. Competing interests The authors declare that they have no competing interests. Authors' contributions EG conceived of the study, participated in the design of the study and drafted the manuscript, BM conceived of the study, participated in the design of the study and coordination, performed the statistical analysis, GA participated in the design of the study and performed the statistical analysis, RU conceived of the study, participated in the design of the study. All authors read and approved the final manuscript. Appendix Sample questions Chronic diseases While working in a health center as a general practitioner, you have noticed that hypertension prevalence is high among the people living in the region under your responsibility. Which of the following would be your choice as primary prevention method? a) I would educate the hypertensive patients on their disease. b) I would treat the hypertensive patients with antihypertensive drugs. c) I would send the hypertensive patients to a secondary care hospital for further investigation and treatment. d) I would educate healthy individuals on risk factors associated with hypertension and prevention methods. Nutritional rules in community Which of the followings is the most common childhood nutritional disorder in Turkey? a) Protein calorie deficiency b) Marasmus c) Iron deficiency anaemia d) Rickets Demography Which of the following is wrong ? a) Demography is a science that analyse the body, structure and differentiations of human populations. b) The goal of family planning is to decrease current number of population. c) Dependent population ratio is found by dividing the total number of population younger than fifteen years and older than 65 years of age by the total number of population between 15–65 years of age. d) Principal of pronatalist population policy is to increase the total number of population. Health Management Which of the followings is not one of the basic records kept in a health center? a) Household determination card. b) Follow-up card for the females between 15–49 years old. c) Follow-up card for aged individuals. d) Antenatal and postnatal follow up card. e) Infant and child follow-up card. Occupational health Which of the followings is not among the responsibilities of an occupational health unit? a) Health prevention services in work settings b) Work safety preventions c) Following up the health and safety conditions in work settings d) Preventing any interruption in production e) Giving outpatient clinic services in work setting. Communicable diseases An 11 year old girl was bitten by a neighbour-dog while she was playing in her house-garden. Which of the followings is not required as an immediate intervention? a) To investigate if the dog is vaccinated. b) To vaccinate the girl for rabies prevention. c) To clean the wound by soap and water. d) To apply one dose of tetanus vaccine. e) To try to understand how the dog bit the girl. Mother and child health Which of the followings is the most common used effective family planning (contraception) methods? a) Intrauterine device b) Withdrawal (coitus interruptus) c) Combined oral contraceptives d) Condom e) Subcutaneous implants Environmental health Which of the followings best represents the environmental health related responsibilities of a general practitioner who works in a health centre? a) Waste control and giving education to correct misapplications b) Analyzing and chlorinating drinking water, control of potable water c) Controlling and improving the condition of toilets, d) Coordination of conduction of above mentioned services by auxiliary personnel of health centre, although these services are among the tasks of municipality. e) All of the statements above are true. Epidemiology After looking over one-year medical records of an internal medicine outpatient clinic, it was found that 25 % of the diagnoses were Diabetes mellitus. Regarding this result a screening procedure was conducted in the field and Diabetes mellitus prevalance was found 5 %. Which of the followings can not be the conclusion of above mentioned situation? I) Outpatient clinic may admit people coming from other regions. II) Outpatient clinic records represent the health status of the community. III) One-fourth of the patients have Diabetes mellitus diagnosis. IV) Field studies are needed to determine the real prevalance of a disease. a) I, II b) I, III c) II, III d) I, II, IV e) II, III, IV Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549539.xml |
554781 | Analysis of leukocyte membrane protein interactions using protein microarrays | Background Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R. Results Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1–40 μg ml -1 corresponding to a limit of sensitivity of 0.01–0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads. Conclusion We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins. | Background Protein-protein interactions are fundamental to biological processes and their analysis is essential for the understanding of cellular pathways. Given the complexity and the dynamic range of the proteome, estimated at 10 7 proteins, the elucidation of protein interactions requires the development of comprehensive, high-throughput proteomic methods that allow quantification of multiple proteins simultaneously [ 1 , 2 ]. The development of protein microarrays represents an attractive new high-throughput technology platform. It involves the printing of ordered arrays of biomolecules onto a solid surface in miniaturized format that allows for the simultaneous determination of multiple interactions using small amounts of samples within a single experiment. The basic principles for highly sensitive "microspot" ligand-binding assays were described by Ekins [ 3 , 4 ] who proposed the "ambient analyte theory" and showed that microspots containing small amounts of capture molecules were able to detect low analyte concentrations with very high accuracy and sensitivity. Since then, miniaturized protein arrays are emerging as one of the most powerful proteomics tools but their application is far more complex [ 5 ] than the DNA microarrays (reviewed in [ 6 - 8 ]) due to structural complexity and heterogeneity of proteins, including their post-translational modifications. Binding of the proteins onto the solid surface of an array must maintain tertiary structure sufficient for functions such as receptor-ligand binding or antibody reactivity. Chemically derivatized microarray surfaces [ 9 , 10 ] or the use of mAb [ 11 , 12 ] have been shown to maintain protein functionality, thus increasing the potential for successful application of microarray technology in proteomics. The study of leukocyte membrane protein interactions provides a particular need because of the large number of interactions yet to be defined [ 13 , 14 ] and a technical challenge as these interactions are often of very low affinity with K D in the range 1–200 μM [ 15 , 16 ]. Although weak, these interactions are important in the context of leukocytes interacting with other cells as illustrated by all the functional data on the interaction of CD8 with MHC Class II (K D = 200 μM) [ 17 ]. The proteins involved usually contain folded domains, the most common type belonging to the immunoglobulin superfamily (IgSF) [ 13 ]. Such domains often interact through large faces of the proteins and require proper folding [ 18 , 19 ]. When measuring low affinity interactions, misleading results can be obtained from unfolded or aggregated materials which are not really a problem when dealing with high affinity interactions such as with cytokines and their receptors, or between proteins and linear epitopes such as lectins and carbohydrates. In addition many leukocyte surface proteins are heavily glycosylated and the oligosaccharides, even if not directly involved in binding, may be important in maintaining biologically active proteins [ 20 ]. Thus, in applying the protein microarray technology to the study of leukocyte surface protein interactions, it is imperative that the proteins are expressed in eukaryotic systems to ensure correct disulphide bond formation and post-translational modifications. In this study we chose a well characterized interaction between CD200 (previously called OX2) and its receptor CD200R (reviewed in [ 21 ]) as a model system to devise a high throughput protein array method for characterization of the interactions between leukocyte surface proteins. CD200 is a widely distributed membrane protein with two extracellular IgSF domains and a short cytoplasmic region unlikely to signal. It interacts with a receptor (CD200R) expressed mostly on myeloid cells, which also has two extracellular IgSF domains but a longer cytoplasmic region with several tyrosine residues that can be phosphorylated [ 22 ]. Functional analysis suggests that the leukocyte CD200 protein can mediate a down-regulatory signal to myeloid cells through the inhibitory CD200R. Thus the CD200 null mice have an increased susceptibility to autoimmune disease induction and myeloid cells expressing CD200R are more activated [ 23 ]. CD200 and a viral homologue found in Kaposi sarcoma virus, when expressed at the cell surface, gave inhibition of production of inflammatory cytokines from activated macrophages [ 24 ]; and targeting the CD200-CD200R interaction with agonistic mAb or CD200-Fc fusion proteins in vivo ameliorates autoimmunity in disease models [ 25 , 26 ]. Protein arrays can be divided into two major classes: 'forward phase' if the analytes are captured from solution; or 'reverse phase' if the analytes are bound directly to the solid phase [ 27 ]. In forward phase protein microarrays, a bait molecule such as an antibody is immobilized onto a solid support to capture the analytes which can be proteins in purified form, or in complex solutions such as cell lysates [ 12 ] or tissue samples [ 27 , 28 ]. The bound analytes are detected either by direct labelling or via a secondary antibody. In reverse phase arrays, the analytes (typically purified proteins or cell lysates) are directly immobilized on the solid phase and antibodies or interacting proteins are applied in solution phase. The analytes can be labelled directly or detected using tags and signal amplification. We have used the forward phase approach in the mapping of antigenic epitopes of hCD200R where different antibodies were immobilized on epoxy coated glass slides, incubated with the hCD200R analyte and detected with fluorescently labelled anti-CD200R antibodies. We have applied reverse phase arrays to three different purposes: -to test the reactivity of the fluorescently labelled mAb with directly immobilized hCD200R protein, -to map epitopes located near the ligand binding site using arrayed mutant hCD200R recombinant proteins and detection with fluorescently labelled mAb that block ligand-receptor interactions and -to detect the low affinity binding of immobilized CD200R to the multivalent CD200 ligand presented on fluorescently labelled beads. Our study extends the use of protein microarrays to the detection of transient cell surface protein interactions, which are of lower affinity than the reported cytokine arrays [ 29 , 30 ]. Results and discussion Quantitative binding of DX147, DX136 and OX108 mAb to human CD200R Purified, soluble recombinant hCD200R protein, engineered with domains 3 and 4 of rat CD4 as an antigenic tag (hCD200R-CD4d3+4) [ 31 ] was directly immobilized at different concentrations on epoxy-coated glass slides, in a reverse phase array as illustrated schematically (Fig. 1A ). The hCD200R array was tested for reactivity with three different mAb: DX147, and two previously reported [ 31 ] mAb DX136 and OX108 able to block ligand binding. Controls on the arrays included recombinant mouse CD200R-CD4d3+4 protein (mCD200R) [ 31 ] and rat CD4d3+4. Figure 1B and 1C illustrate the strong and specific binding of all three fluorescently labelled mAb to hCD200R, as demonstrated by minimal reactivity with rCD4 (rat CD4d3+4) and lack of cross-reaction with mCD200R. DX147 gave the strongest labelling with linear binding from 0.08 to 20 μg ml -1 mAb, reaching the upper limits of detection under the optimized voltage settings (65,000 units of green fluorescence) at 40 μg/ml (Fig. 1B ). Binding of DX136 was linear over the full range of concentrations with a maximum binding of 48,000 units. OX108 bound more weakly, reaching a maximum value of 32,000 units. Sensitivity of detection, defined as two-fold binding above background, was estimated as the lowest hCD200R-CD4d3+4 concentration tested (0.08 μg ml -1 ) for DX136. A three-fold signal to noise ratio was achieved for DX147 at that concentration suggesting that sensitivity of detection was closer to 0.05 μg ml -1 . For OX108, the limit of sensitivity was estimated at 0.3 μg ml -1 . Thus DX147, DX136 and OX108 were able to detect 0.5 pg, 0.8 pg and 3.0 pg of hCD200R per spot respectively estimating a spot volume of 10 nl. The limit of sensitivity achieved was therefore between 8 and 50 attomol, assuming a molecular weight of 60,000 for hCD200R-CD4d3+4 protein. The amounts of human and mouse CD200R-CD4d3+4 and rCD4d3+4 protein on the microarray spots were similar as visualized by the red fluorescence of OX68 mAb recognising the CD4 tag present in each of the recombinant proteins (Fig. 1B and 1D ). This indicates that the amount of protein detected is proportional to the amount arrayed in each spot and is highly reproducible. The limit of sensitivity of protein detection with Alexa 647-OX68 was approximately 0.3 μg ml -1 , corresponding to 50 attomol of CD200R-CD4d3+4 proteins. Although not all molecules will be in a proper orientation for equal access to both anti-CD200R and anti-CD4, as illustrated in Fig. 1A , our results suggest that on average, there is a good correlation between the amount of specific antibody bound and the amount of protein arrayed. Figure 1 Quantitative binding of mAb to hCD200R-CD4d3+4. (A). Scheme illustrating a reverse phase microarray in which purified CD200R proteins were immobilized and then screened with fluorescent mAb specific for hCD200R or for the antigenic rCD4 tag (OX68) of the hybrid recombinant protein.(B). Typical microarray shows binding of OX68 mAb (red) to control proteins or the overlapping binding of hCD200R mAb and OX68 (yellow) to immobilized human CD200R.(C). Shows the green fluorescence intensity of each spot for all four replicates (mean ± SEM).(D). Shows red fluorescence intensity due to binding of OX68 mAb using data in left panel for DX147 (similar levels were found with the other mAb). Serial two-fold dilutions of purified, soluble, recombinant human and mouse CD200R-CD4d3+4 proteins, and of control rat CD4d3+4 were arrayed onto epoxy-coated microscope slides. Each protein dilution series was arrayed in 3 rows of 4 spots, ranging in concentration from 40 μg ml -1 (first spot) to 0.08 μg ml -1 (spot 10), with control spotting buffer containing 0.5 mg ml -1 BSA in the last two spots. All arrays were performed in quadruplicate and a representative set is shown in (B). Each slide was incubated for 16 h at 4°C with a mixture of hCD200R mAb (DX147, DX136 or OX108) labelled with Alexa-555 (indicated as green fluorescence measured at 532 nm) and rCD4 mAb (OX68, detecting the antigenic tag and allowing for measurement of recombinant protein concentration) labelled with Alexa-647 (red fluorescence measured at 635 nm). At the highest concentrations, the hCD200R spots appear either white (saturating conditions) or yellow, due to the combination of green and red signals given by the specific binding of the Alexa-555-mAb to hCD200R and Alexa-647-OX68 mAb respectively. Quantitative measurements are expressed as mean fluorescence units at 532 nm (green) and 647 nm (red) versus amount of protein arrayed. Orientation via antibody immobilization for epitope mapping on human CD200R We used forward phase protein microarrays to define the epitopes of hCD200R recognized by the three mAb introduced in the previous section. Serial dilutions of DX147, DX136 and OX108 mAb and the control CD4 mAb OX68 were directly immobilized on epoxy-coated glass slides as shown (Fig. 2A ). The mAb arrayed act as capture reagents for hCD200R, used at a concentration of 20 μg ml -1 . Each capture mAb binds to a different epitope on the hCD200R-rCD4d3+4 recombinant protein, thus orientating the protein on the array in a conformation that permits or restricts access to the same panel of mAb, used as detection reagents (Fig. 2A ). Visual observation (Fig. 2B ) and quantitative analysis (Fig. 2C ) showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. As expected, OX68 is the most suitable capture mAb, as it binds the common CD4 tag allowing exposure of the two extracellular domains of hCD200R and binding of all three specific mAb. When the detection mAb is the same as the capture mAb, no significant binding above background is observed (Fig. 2C ). DX147 binds specifically to hCD200R-CD4d3+4 orientated via OX68 and DX136 (16,021 and 12,570 green fluorescence units respectively at 80 μg ml -1 of capture mAb), but not via OX108 or via itself. This is an indication that the binding epitopes for DX136 and DX147 are dissimilar. DX136 in turn, binds well to hCD200R-CD4d3+4 immobilized on OX68 (13,954 units of green fluorescence at 80 μg ml -1 ) and to a lesser degree to hCD200R-CD4d3+4 immobilized on DX147 (2,647 units), while not at all to hCD200R captured by OX108 (496 units). These data indicate that the DX136 epitope is spatially distinct from the DX147 epitope, but in close proximity to the OX108 epitope. This conclusion is substantiated by the lack of binding of OX108 mAb to hCD200R-CD4d3+4 captured on DX136. Orientating hCD200R via mAb OX68 allows for specific binding of all three anti-human CD200R mAb, in a linear fashion with limits of sensitivity of about 5 μg ml -1 of immobilized mAb. The maximum amount of signal was obtained by capturing hCD200R with 80 μg ml -1 of mAb OX68 and was equivalent to that observed by directly immobilizing approximately 10 μg ml -1 hCD200R. OX68 is therefore the best mAb for capturing the chimaeric hCD200R-CD4 protein for optimal detection of hCD200R epitopes. Figure 2 Analysis of mAb reactivity with hCD200R by orientation via antibody immobilization. (A). Scheme of one forward phase microarray in which purified human CD200R protein was immobilized via OX68 mAb and detected with the DX136 hCD200R mAb (green fluorescence).(B). Typical microarray shows binding of the hCD200R mAb (green) and OX68 mAb (red) to hCD200R immobilized via four different capture mAb.(C). Shows the mean fluorescence intensity ± SEM for each spot of all replicates. Serial two-fold dilutions of capture human CD200R mAb OX108, DX136 and DX147 and control rat CD4 mAb OX68 were arrayed onto epoxy-coated microscope slides. Each mAb dilution series was arrayed in quadruplicate of 2 rows of 6 spots, ranging in concentration from 80 μg ml -1 (first spot) to 0.16 μg ml -1 (spot 10), with control spotting buffer containing 0.5 mg ml -1 BSA in the last two spots. The whole array was repeated on the slide for a total of 8 replicates per spot. Each slide was incubated for 2 h with 20 μg ml -1 of purified recombinant hCD200R-CD4d3+4 protein, prior to incubation with Alexa-555-labeled CD200R mAb (DX147, DX136 or OX108) or Alexa-647 control rCD4 mAb (OX68). Quantitative measurements are expressed as mean fluorescence units at 532 nm (green) and 635 nm (red) versus amount of capture mAb arrayed. Mapping of CD200R antigenic epitopes using mutants Both CD200R and its ligand, CD200 contain two extracellular IgSF domains. The ligand-receptor interaction is therefore likely to occur in an end-to-end topology, requiring opposing cell surfaces to come into close proximity [ 22 ]. Previous studies have shown that the membrane distal N-terminal domain of CD200 is involved in binding its receptor [ 32 ]. In a recently published study [ 33 ], site directed mutagenesis was employed to map the ligand-binding domain of human CD200R using the structure of a typical Ig V domain, that of the human junctional adhesion molecule 1, JAM1 to predict the positions of out-pointing residues [ 34 ]. A panel of mutants was designed so as to target residues likely to be out-pointing from predictions of the beta strands of the N-terminal IgSF domain of hCD200R. The binding sites of CD200 and of the OX108 mAb known to block ligand interaction to the hCD200R mutants, were shown to be on the GFCC' face of the N-terminal IgSF domain [ 33 ]. The same panel of hCD200R-CD4d3+4 mutant proteins (Table 1 ) was analyzed by reverse protein microarrays (analogous to Fig. 1A ) for binding to mAb DX147, DX136 and OX108 in order to map these epitopes. The mutant proteins were expressed by transient transfection in serum-free medium, concentrated and arrayed. Purified human and murine CD200R serving as positive and negative controls were immobilized at concentrations ranging from 0.08 to 40 μg ml -1 . Lack of binding by a specific mutant or group of mutants is suggestive of the corresponding residues defining the location of the antigenic epitopes. None of the hCD200R mutants tested had lost binding to DX147, confirming unpublished data that this epitope lies, not in the N-terminal domain of CD200R but in the membrane proximal domain. Some of the mutants showed increased binding of mAb over the wild type despite these being normalized with OX68 (e.g. R67, I71K). This presumably reflects variations in epitope availability due to direct immobilization of the mutant proteins on the array, which are less likely to occur in BIAcore studies where mutants were immobilized via OX68 mAb [ 33 ]. Thus our data analysis was focused on mutants with major impairment in binding activity. Nine of the mutants showed reduced binding to either DX136 and/or OX108 mAb, relative to wild type hCD200R, as represented graphically in Figure 3 . One mutant illustrated (E75K) did not affect binding in a significant manner. The position of these mutants is shown on the model of the N-terminal domain of hCD200R, based on a typical IgSF domain of similar size (Fig. 4 ). Panel A shows the mutants affecting CD200 binding, as reported in [ 33 ]. These mutants were mostly in the GFCC' face and in particular the F and C strands. Panel B shows the mutants with reduced binding to DX136 and/or OX108. It is immediately noticeable that mutants within the F and C strands also appear to have lost binding to DX136 and OX108 mAb, suggesting close proximity of these epitopes to the CD200 binding site, in agreement with the ligand blocking activity of these mAb. However, the key residues were not identical as shown by the differing effects of the mutants I71K and R67D. Furthermore, the microarray study confirms the results obtained by individual analysis of each mutant by surface plasmon resonance in terms of residues involved in OX108 binding; the most critical amino acid appears to be R67 located in the B-C loop [ 33 ]. The finding of mutants that had apparently gained CD200R mAb binding activity compared to the OX68 mAb recognising the CD4 tag, shows that spotting can have effects not seen by indirect methods such as the BIAcore. However there was very good correlation between available BIAcore data and microarray data. Thus this method provides a rapid high throughput method to identify the rare mutants that affect antigenic activity that can then be characterised further e.g. by BIAcore analysis. Table 1 hCD200R protein mutants tested. Mutant proteins were constructed as described in [33], expressed by transient transfection in serum-free medium, concentrated and immobilized on epoxy-derivatized slides. The predicted positions of the residues are located in the modelled V-like N-terminal IgSF domain unless noted otherwise (C domain). # Mutant Name Predicted Position 1 Wild-type 2 D30K* N-term 3 K40D* A strand 4 L42E* A strand 5 E44K* A strand 6 E44A A strand 7 E44D* A strand 8 M53K A-B loop 9 N56D B strand 10 P62F B strand 11 I64S* B-C loop 12 R67D B-C loop 13 I71K C strand 14 T73R C strand 15 E75K C strand 16 R79E C-C' loop 17 Q81K C-C' 18 S83D* C" strand 19 E97K C" strand 20 T106D* D strand 21 D116K* D-E loop/ E strand 22 A123D E strand 23 Y129D F strand 24 R131E F strand 25 I133K F strand 26 D138K* F-G loop 27 R143D* F-G loop 28 H146D G strand 29 Q148E* G strand 30 L150D* G strand 31 T156N* A strand C domain 32 N160D A-B loop C domain 33 A175D* B-C loop C domain * Mutants expressed with concentration below sensitivity threshold, for which no antibody binding data could be derived. Figure 3 Mapping of antigenic epitopes on hCD200R mutants. The hCD200R mutants described in Table 1 and produced by transient transfection were arrayed (reverse phase) and tested for specific binding to mAb DX147, DX136 and OX108 and for reactivity with OX68 to quantify the relative amount of protein in each sample as described in Methods. Results for a panel of 10 mutants are plotted as percent antibody binding normalized to the wild type, non-mutated hCD200R protein (WT) values. Figure 4 Mapping of DX136 and OX108 epitopes on the N-terminal domain of human CD200R. (A). The mutants giving complete or nearly complete inhibition of CD200 binding, determined by BIAcore analysis, are indicated with red circles (<35% binding compared to non-mutated WT hCD200R protein), whereas those giving partial effects (35–70% binding) are depicted in orange. Data from [33].(B). The mutants giving severe inhibition (<35% compared to WT) of OX108 and DX136 mAb binding, as determined by microarray analysis, are depicted in blue and yellow respectively. Dark green circles represent mutants that severely impaired both OX108 and DX136 mAb binding. Mutants partially affecting DX136 binding (35–50%) are shown in pale yellow. Mutants that severely affect DX136 binding (35% binding or less) but have only a partial effect on OX108 binding (50%) are represented in pale green. Open circles depict mutants that did not affect the binding of CD200, OX108 or DX136 mAb. The CD200R model is based on a typical Ig V domain from the human junctional adhesion molecule-1 (JAM1) [34]. The beta sheets are labelled with the GFC face orientated in front and the BED face behind. Reactivity of human, rat and mouse CD200R with multimeric human CD200 The interaction of human CD200 with its receptor hCD200R is of low affinity, with a K D of ~0.5 μM at 37°C and t 1/2 of 7 s [ 24 ], typical of the interaction of many leukocyte membrane proteins [ 15 ]. Such an interaction could not be detected when immobilized hCD200R was incubated with fluorescently labelled purified monomeric hCD200 protein (data not shown). In order to develop leukocyte membrane receptor-ligand microarray assays, high avidity detection reagents are required. Recombinant hCD200 protein was constructed by linking the extracellular domains of human CD200 with domains 3 and 4 of rat CD4 (CD4d3+4) as an antigenic tag. This construct contains a 19 amino acid sequence at the C-terminus of the protein, which can be enzymatically biotinylated on a specific lysine residue using the E. coli BirA enzyme [ 35 ]. Expression of the construct was demonstrated by inhibition of a rat CD4 ELISA using OX68 mAb. The recombinant protein bound the mAb OX104 (mouse anti-human CD200), indicating that it was antigenically active, as assessed by BIAcore analysis (data not shown) and its biotinylation was confirmed by streptavidin binding. The biotinylated hCD200-CD4d3+4 protein or the control CD4d3+4 protein were attached to avidin-coated FITC-fluorescent beads via their biotin tag, thus creating polyvalent CD200 and control reagents. These beads were used to detect specific binding of CD200 to human, mouse and rat CD200R proteins arrayed directly on epoxy-coated glass slides (Fig. 5A ). Additional controls included rCD4d3+4 protein arrayed on the slide and tested for reactivity with both types of beads. Strong binding of hCD200-beads to all three CD200R proteins was observed (Fig. 5B and 5C ) indicating that the proteins immobilized on the glass surface had retained their capacity to bind ligand with maximum mean values of 63,500 green fluorescence units for rat CD200R (saturating), 43,600 for mouse CD200R and 23,800 for human CD200R. The fluorescence appears granular as one is actually visualizing the small fluorescent beads. Binding was detected at concentrations of receptors ranging from approximately 5 to 40 μg ml -1 corresponding to 1–8 femtomol per spot on the microarray. The non-specific binding of hCD200-beads to immobilized rCD4 protein was negligible (Fig. 5B and 5C ) and control CD4d3+4 beads did not react with any of the arrayed proteins (data not shown). The multivalent hCD200-beads cross-reacted with rat and mouse CD200R as expected, as BIAcore analysis has shown that hCD200 interacts with human, rat and mouse CD200R with affinity constants within a log of each other [ 31 ]. Sensitivity of detection, defined as two-fold binding above background was achieved with concentrations of 5 μg ml -1 for rat CD200R and 10 μg ml -1 for human and murine CD200R. This corresponds to 1–2 femtomol of immobilized receptors interacting with the multimeric human CD200 ligand. Figure 5 Binding of multimeric CD200 ligands to CD200R proteins. (A). Diagram of the reverse phase array depicting immobilized CD200R interacting with the multivalent bead ligand.(B). A representative microarray set showing binding of hCD200 beads to CD200R-CD4d3+4 proteins, but not control rat CD4.(C). Mean fluorescence intensity ± SEM of all four sets is shown versus receptor protein concentration arrayed. Serial two-fold dilutions of purified, soluble, recombinant human, mouse and rat CD200R-CD4d3+4 proteins, and of control rat CD4 were arrayed onto epoxy-coated microscope slides. Each receptor protein dilution series was arrayed in 3 rows of 4 spots, ranging in concentration from 40 μg ml -1 (first spot) to 0.08 μg ml -1 (spot 10), with control spotting buffer containing 0.5 mg ml -1 BSA in the last two spots. Only the first 8 dilutions (2 rows) are shown in (B) and analyzed in (C). All arrays were performed in quadruplicate. All receptors were arrayed on the same slide, which was incubated for 16 h at 4°C with polyvalent human CD200-CD4d3+4 FITC-fluorescent beads. At the highest concentrations, the hCD200R spots appear white (saturating conditions). Quantitative measurements are expressed as fluorescence units at 532 nm (green) versus amount of protein arrayed. Conclusion In order to study interactions of leukocyte membrane proteins using high throughput microarray techniques, it was essential that the proteins be immobilized at low concentrations and in a biologically active form. It is critical that weak interactions between leukocyte membrane proteins be detected without interference by the anomalous binding due to denatured proteins, which is more of a contributing factor in the study of low affinity interactions. We first established that recombinant CD200R proteins could be immobilized directly in reverse phase arrays, in a conformation capable of reacting with three different mAb. We then demonstrated that recombinant mutant hCD200R proteins produced in transient expression systems were present in sufficient amounts to be immobilized directly and tested for reactivity with specific mAb, permitting mapping of epitopes. These data show that high throughput analysis of cell surface proteins can be achieved in reverse phase arrays using recombinant proteins derived from transient transfectants in a non-purified form. We also used forward phase arrays for competitive analysis of antibodies and mapping of their epitopes. This approach is valuable for rapidly screening antibody specificities and assessing protein orientation needed for optimal presentation of immunogenic determinants. We also showed that binding of CD200 ligand to its cell surface receptor can be achieved by increasing the avidity of the reaction via coupling of the biotinylated recombinant CD200 protein to fluorescently labelled avidin coated beads. The fluorescent beads offer an efficient technology for the analysis of low affinity interactions typical of those observed for leukocyte membrane proteins and many other cellular proteins. Methods Materials Monoclonal antibodies (mAb) DX147 (rat IgG1), and DX136 (rat IgG2a) to human CD200R were generously given by DNAX Research Institute (Palo Alto, CA). The mAb OX108 (mouse IgG1) to human CD200R [ 31 ] and OX68 (mouse IgG1) to rat CD4 domains 3 and 4 (rCD4d3+4) have been described previously [ 36 ]. Recombinant proteins The soluble biotinylated forms of human, mouse and rat CD200R were produced as described [ 22 , 31 , 36 ]. Briefly, the entire extracellular region of human, mouse or rat CD200R was amplified by PCR and cloned in the pEF-BOS-CD4d3+4bio-XB vector [ 35 ]. These constructs were then subcloned into the expression vector pEE14, and stably secreting CHO.K1 cell lines were established [ 37 ]. Human, mouse or rat CD200R-CD4d3+4 proteins were purified from the tissue culture medium by immunoaffinity chromatography with OX68 mAb-Sepharose 4B that recognizes the CD4 protein tag [ 36 ]. Prior to use, the purified CD200R proteins were fractionated by gel filtration on Superdex S-200 (Pharmacia, Uppsala, Sweden) to exclude protein aggregates. The soluble, biotinylated form of human CD200 was produced in an identical fashion, by subcloning the amplified extracellular region of human CD200 [ 38 ] using Xba I/ Sal I digestion to the pEF-BOS-CD4d3+4bio-XB vector [ 36 ]. This construct was used to transfect HEK293T cells using the calcium phosphate method. The protein expressed was enzymatically biotinylated and used to generate multivalent binding reagents by coupling to avidin-coated fluorescein isothiocyanate (FITC)-loaded beads (Spherotech Inc., Libertyville, IL) as described previously [ 35 ]. Mutants of human CD200R (hCD200R) were prepared as described [ 33 ]. Briefly, the mutations were introduced by site directed mutagenesis using PCR and two mutagenic oligonucleotides into a construct comprising the extracellular domains of human CD200R together with domains 3 and 4 from rat CD4 (CD4d3+4) as an antigenic tag. The mutants were transiently expressed in HEK 293T cells using X-VIVO 10 media (BioWhittaker, Nottingham), concentrated about 10 fold and levels of expressed protein quantified by ELISA. This media contains 1 mg ml -1 BSA so after concentration the final protein concentration is around 10 mg/ml. Antibody labelling Purified antibodies were dialysed against PBS prior to labelling with Alexa Fluor 555 or Alexa Fluor 647 fluorescent amine-reactive dyes using the Molecular Probes Monoclonal Antibody Labelling Kits (Cat. No. A-20186 and A-20187) and according to the manufacturer's instructions (Molecular Probes, Invitrogen Ltd.). Labelling reactions were carried out using 100 μg of IgG and yielded labelled proteins ranging in concentration from 1 to 4 × 10 -6 M. The degree of conjugation was estimated at 2–4 moles of dye per mole protein. Labelled antibodies were stable for up to 2 months at 4°C. Preparation of microarrays Protein solutions to be arrayed were prepared in 96 well plates and 12 μl aliquots were transferred to single wells of Genetix 7020, 384-well plates (Genetix Ltd, New Milton, UK). Concentrations tested ranged from 0–80 μg/ml and all dilutions were performed in Protein Array Spotting Solution (Genetix) with the addition of 0.5 mg/ml BSA and 0.02% NaN 3 . A QArrayMini microarray printer (Genetix) was used to apply the protein solutions onto epoxy-coated microscope slides using 300 μm solid tipped tungsten microarraying pins (Genetix). Preliminary experiments established the printing conditions with fluorescently labeled OX68 mAb. Of several types of slides tested, the epoxy-coated ones were the best in terms of spot morphology, cost and reproducibility and were used in all subsequent experiments. Most array designs were performed using 8 pins to obtain spots with a 440 μm diameter and centre-to-centre spot spacing of 700 μm in both directions. Source plates were kept at 8°C, and a 65% average internal humidity was maintained. After printing, the slides were left in the arraying chamber for 30–60 minutes under the same conditions. The slides were then washed using the Protein Array Processing Kit (Genetix Ltd; stored at 4°C and the solutions supplemented with 0.02 % NaN3) by inversion for 1 min in Clean Up Buffer (Genetix) to remove unbound proteins and incubation for 30 min in Blocking Buffer. Slides were washed 3 times in PBS, once in H 2 O to remove excess salt and dried using an air brush, and stored at 4°C, with desiccant in a sealed slide box. Preliminary experiments were done by forward phase arrays to establish optimal conditions. OX68 mAb (100 μg ml -1 to 20 μg ml -1 ) was immobilized, incubated with rat CD4 at 5 μg ml -1 0.1 mg ml -1 BSA, washed and incubated with labeled W3/25 mAb (5 μg/ml) and linearity of detection demonstrated. Specificity was also shown by the fact that OX68 was not reactive with CD4 immobilised on OX68 and vice versa. Labelling of microarrays Slides were placed in hybridization chambers (Corning Incorporated, UK) and the humidification wells filled. LifterSlips, (Erie Scientific, Portsmouth, USA) were placed gently over the marked boundaries of the arrays and the binding reagent (25–70 μl) was introduced with a micropipette. In experiments where CD200R-CD4 hybrid proteins (including the mutant studies) were arrayed, Alexa-555 anti-CD200R antibodies (mAb DX147, DX136 or OX108; 5–10 μg ml -1 ) were added to measure the amount of specific antibody bound and Alexa-647-CD4 mAb (OX68 5–10 μg ml -1 ) was included to assess the amount of hybrid protein present. In experiments where capture antibodies were arrayed (Fig. 2 ), a 2 h incubation with purified protein, such as CD200R-CD4d3+4 or CD4d3+4 at 20 μg ml -1 , was performed prior to incubation with the detection antibodies. In the experiments detecting CD200 (ligand) binding to immobilized CD200R (Fig. 5 ), arrays were incubated with biotinylated hCD200-CD4d3+4 streptavidin-FITC beads. Incubations with detection reagents were carried out for 16 h at 4°C unless otherwise stated. The slides were immersed upside down in PBS/0.05% Tween-20, washed thrice with copious amounts of PBS/Tween-20, alternating shaking up and down under liquid in the Copeland jar and gentle rocking for 5–10 min each, followed by PBS (twice for 5 min) and a final H 2 O rinse. All washes were at room temperature and repeated following each incubation. After drying the slides with an air brush, the arrays were scanned using a GenePix4000B microarray scanner (Axon Laboratories, Palo Alto, CA) scanner using 532 nm and 635 nm lasers using the GenePix Pro 5.0 (Axon Laboratories) software. The PMT values were 720 and 1000 (532 nm and 635 nm respectively) for Figure 1 , 780 and 950 for Figure 2 , 900 and 1000 for Figure 3 and 850 for Figure 5 (532 nm only). Data analysis All samples were tested in quadruplicate and all experiments repeated several times. The amount of antibody or ligand bound to the arrayed proteins and the amount of protein present in each spot were determined by comparing the fluorescence intensities read at 532 and 635 nm. Extraction of spot intensity data was performed using GenePix Pro 5.0 (Axon Laboratories) and ScanArray Express (Perkin Elmer). The background, calculated as the median of pixel intensities from the local area around each spot, was subtracted from the mean pixel intensity within each spot. To graphically represent the data, the values of the background-subtracted signal intensities were plotted against the known concentration of the protein spotted in the array. Sensitivity of detection for each spot was defined as a signal to noise ratio (S/N) of two-fold above background. S/N was calculated as: S/N = (background-subtracted median signal intensity) / (standard deviation of background signal intensity). In the case of the mutant hCD200R proteins generated from culture supernatants of transient transfections, where protein concentration is unknown, the background-subtracted values for both 532 and 635 nm-signal intensities were corrected for internal protein signal by subtracting the corresponding value of a "mock transfectant" spot. The corrected values for the red channel (representing the amount of protein assessed from the CD4 content) were normalised to 100% with respect to the wild-type hCD200R transient transfection sample. All hCD200R mutants with red channel values below 50% were assumed to contain insufficient amount of protein and were excluded from the analysis. The green channel background-subtracted, "mock"- transfectant corrected values (G) were divided by the corresponding red channel ones (R) to correct for variations in the amount of expressed protein contained in each individual spot (G/R ratio). Finally, the G/R ratio was normalized to 100% with respect to the hCD200R-CD4d3+4 wild-type protein, before graphical representation. List of abbreviations IgSF, immunoglobulin superfamily; hCD200, human CD200; CD200-CD4d3+4, chimaeric recombinant CD200 protein with rat CD4 domains 3+4; hCD200R, human CD200 receptor; mCD200R, mouse CD200 receptor; rCD200R, rat CD200 receptor; SPR, surface plasmon resonance; WT, wild type. Authors' contributions ML set up and developed the protein arrays with DV specialising in the experiments with the multimeric beads and data analysis. Both wrote the paper and DV prepared the figures. DH produced the mutant CD200R proteins and advised on their analysis. MF produced the mAb OX108 and the human, mouse and rat CD200R proteins. NS provided expertise in setting up the system for protein microarrays and their basic analysis. NB devised the initial project, advised throughout and helped with the manuscript writing. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554781.xml |
524500 | Gene expression profiling of epithelial ovarian tumours correlated with malignant potential | Background Epithelial ovarian tumours exhibit a range of malignant potential, presenting distinct clinical phenotypes. Improved knowledge of gene expression changes and functional pathways associated with these clinical phenotypes may lead to new treatment targets, markers for early detection and a better understanding of disease progression. Results Gene expression profiling (Affymetrix, U95Av2) was carried out on 18 ovarian tumours including benign adenomas, borderline adenocarcinomas of low malignant potential and malignant adenocarcinomas. Clustering the expression profiles of samples from patients not treated with chemotherapy prior to surgery effectively classified 92% of samples into their proper histopathological group. Some cancer samples from patients treated with chemotherapy prior to surgery clustered with the benign adenomas. Chemotherapy patients whose tumours exhibited benign-like expression patterns remained disease free for the duration of this study as indicated by continued normal serum CA-125 levels. Statistical analysis identified 163 differentially expressed genes: 61 genes under-expressed in cancer and 102 genes over-expressed in cancer. Profiling the functional categories of co-ordinately expressed genes within this list revealed significant correlation between increased malignant potential and loss of both IGF binding proteins and cell adhesion molecules. Interestingly, in several instances co-ordinately expressed genes sharing biological function also shared chromosomal location. Conclusion Our findings indicate that gene expression profiling can reliably distinguish between benign and malignant ovarian tumours. Expression profiles of samples from patients pre-treated with chemotherapy may be useful in predicting disease free survival and the likelihood of recurrence. Loss of expression of IGF binding proteins as well as specific cell adhesion molecules may be a significant mechanism of disease progression in ovarian cancer. Expression levels in borderline tumours were intermediate between benign adenomas and malignant adenocarcinomas for a significant portion of the differentially expressed genes, suggesting that borderline tumours are a transitional state between benign and malignant tumours. Finally, genes displaying coordinated changes in gene expression were often genetically linked, suggesting that changes in expression for these genes are the consequence of regional duplications, deletions or epigenetic events. | Background Epithelial ovarian cancer is the fifth leading cause of death for women in the United States [ 1 ]. Although early stage ovarian cancer can be effectively treated, symptoms of early disease are sufficiently vague that accurate diagnosis is often delayed until the cancer has progressed into more advanced stages [ 2 ]. Treatment of early staged tumours (I through IIa) is associated with a 5-year survival rate of approximately 95% while survival rates drop to less than 30% when diagnosis is delayed until later stages (stage IIb through IV). To improve these statistics, effective early diagnosis and treatment strategies must be developed. Further knowledge of the genes and gene functional pathways involved in ovarian cancer are needed in order to develop these strategies. Microarray technology has revolutionised the study of gene function by providing "snapshots" of global gene expression patterns from different normal and diseased tissues over multiple stages of development. Nowhere has the impact of this technology been more pronounced than in the field of cancer biology where gene expression profiling has been successfully used to objectively classify tumours and, in some instances, identify novel tumour sub-types [ 3 ]. Microarray analyses have also been instrumental in the elucidation of new biological pathways that may be involved in tumour development, as well as, in the identification of new biomarkers of the disease and potential targets of therapeutic intervention. Previous microarray studies of ovarian cancers have focused on the characterisation of differences between normal ovarian epithelial cells (and cell lines) and various types and stages of ovarian tumours [ 4 - 10 ]. In this study, we focus on characterising differences between benign adenomas, borderline tumours of low malignant potential and malignant adenocarcinomas in order to identify changes associated with the acquisition of malignancy and to avoid the technical difficulties associated with obtaining sufficient amounts of normal ovarian surface epithelium. The ovarian tumour tissue samples used in these microarray studies were chosen to accurately represent the range of malignant potential observed clinically. We report here the results of applying clustering and statistical analyses to the microarray expression profiles of 18 ovarian tumours. Our findings indicate that gene expression profiling distinguished properly classify 92% of tumours in this study as benign or malignant. Samples taken from ovarian cancer patients who had been treated with chemotherapy prior to surgery were found not to cluster as a distinct group but rather with either the benign or malignant (not pre-treated) tumours. Chemotherapy patients whose tumours clustered with the benign group remained disease free for the duration of the study as evidenced by continued normal serum CA-125 levels. Profiling the functional categories of co-ordinately expressed genes revealed significant correlation between increased malignant potential and loss of IGF binding proteins, and cell adhesion molecules. In several instances co-ordinately expressed genes sharing functional categories also correlated with chromosomal location. Results Unsupervised clustering of gene expression profiles can reliably identify ovarian tumour types To determine if gene expression profiling can distinguish between histologically determined tumour types, we analysed the profiles of 13 ovarian tumours (Table 1 ) by performing clustering using self-organising maps (SOM) and unsupervised hierarchical clustering (UHC). Self-organising maps are a type of mathematical cluster analysis used to recognise and classify features in complex multi-dimensional data [ 11 ]. SOMs group samples into a user-defined number of clusters based on the similarity of the gene expression profiles. The set of thirteen samples was comprised of four benign adenomas (a_64, a_77, a_97, a_159), four borderline tumours of low malignant potential (b_15, b_65, b_72, b_120) and five malignant adenocarcinomas (c_2, c_4, c_23, c_66, c_79). Analysing all 12,590 probe set values from the 13 samples into four groups resulted in 92% of the samples being grouped into clusters consistent with their histopathological classification (Figure 1a ). One cluster (cluster 0) contained only adenocarcinomas, two clusters (clusters 1 and 2) contained only borderline tumours, and one cluster (cluster 3) contained all of the benign adenomas and one adenocarcinoma sample. In addition, the UHC of the entire data set (Figure 1b ) produced essentially the same clusters as determined by SOM. The only difference between the SOM and UHC results was the stratification of borderline tumours, which are known to be a heterogeneous group of tumours. The SOM clustered the four borderline samples into one group of three borderlines (b_65, b_15, b_72) and one solitary sample (b_120) (Figure 1a ). However, the UHC clustered the four borderline samples into one group containing b_15 and b_120, and one group containing b_65, and b_72. Since c_79 was consistently misclassified, a second tissue sample of c_79 was analysed by microarray and clustered as above. This independently obtained expression profile for c_79 produced the same results. Table 1 Tissue Sample Information Tumor ID Malignant Potential Available Histological Information Stage Chemo a_64 benign Serous cystadenofibroma - - a_77 benign Serous cystadenofibroma - - a_97 benign Serous cystadenoma - - a_159 benign Serous cystadenofibroma - - b_15 low/borderline Serous papillary adenocarcinoma III - b_65 low/borderline Mucinous adenocarcinoma II - b_72 low/borderline Mucinous adenocarcinoma I - b_120 low/borderline Serous papillary adenocarcinoma II - c_2 invasive malignant Serous papillary adenocarcinoma IIb - c_4 invasive malignant Serous papillary adenocarcinoma III - c_23 invasive malignant Serous papillary adenocarcinoma IIIa - c_66 invasive malignant Serous papillary/endometroid adenocarcinoma IV - c_79 invasive malignant Serous papillary carcinoma III - cc_9 invasive malignant Serous papillary carcinoma III Yes* cc_29 invasive malignant Serous papillary carcinoma III Yes # cc_36 invasive malignant Serous papillary adenocarcinoma IIIc Yes* cc_76 invasive malignant Serous papillary adenocarcinoma IIIa Yes* cc_94 invasive malignant Serous carcinoma III Yes* * Taxol/Carbo 3X prior to surgery #Taxol/Carbo 4X prior to surgery Figure 1 Cluster analysis of ovarian tumour expression profiles. Gene expression profiles were obtained from eighteen ovarian tumours. The profiles were analysed by clustering methods in several groups: (a) self organizing map of he thirteen patients not receiving chemotherapy prior to tissue collection, (b) hierarchical clustering of the same 13 patients, (d) self organizing maps of all eighteen patients and (e) hierarchical clustering of all 18 patients. Marker analysis (c) identified the top ten gene most highly correlated with clusters resulting from (a) and (b). Since many of the genes in our data set display no differential expression across the 13 tumours, their contribution to the SOM is negligible and can be considered noise. Removing genes whose expression pattern displayed insignificant variation (low standard deviation) across all samples, we reduced the data set to1000 probe sets. After removing probe sets representing the same gene, the reduced data set contained expression values representing 700 genes. The SOM and UHC of the reduced data set yielded identical clusters to those obtained using the entire data set (Figure 1a and 1b ). To determine the genes most highly correlated with each cluster identified by the SOM analysis, we performed a marker analysis (Figure 1c ) on the reduced set of 700 genes. Marker analysis helps the user discover which genes are most closely correlated with a cluster and provides a measure of how significant that correlation is for each gene. Marker analysis measures the contribution of each gene to the SOM groupings based on a signal to noise ratio calculated from the difference in each gene's mean expression scaled by the sum of the standard deviations across all samples. To avoid having one cluster containing only one sample in the marker analysis, we grouped clusters cluster 1 and cluster 2 containing the borderline samples together, creating three clusters (Figure 1c ) consisting of the benign adenomas and c79 (SOM_a), borderline adenocarcinomas (SOM_b), and the malignant adenocarcinomas (SOM_c). Genes highly correlated with each SOM group were expressed strongly in the tumour type associated with that SOM group and poorly expressed in the other SOM groups. It is interesting to note that the 10 genes highly correlated with SOM_a were expressed at intermediate levels in borderline tumours. Similarly, the 10 genes highly correlated with SOM_b were expressed at intermediate levels in the adenocarcinomas of SOM_c. Gene expression profiles are correlated with recurrence Five of the cancer patients in our study were treated with chemotherapy prior to surgery. We added the microarray profiles of these patient samples to our analysis in order to determine if they would cluster into a new distinct group or into one or more of the existing groups. The SOM (Figure 1d ) and UHC (Figure 1e ) clusters resulting from the analysis of all data (12,590 expression values) from all eighteen samples into four clusters differ only in the stratification of the borderline samples. The addition of the five samples from patients who received chemotherapy prior to surgery did not change the cluster assignments of the original thirteen samples. Clustering of the reduced set of 700 genes (see above), resulted in the same patterns of clustering as determined using the entire set of 12,590 expression values (Figure 1d and 1e ). Interestingly, the five samples from patients pre-treated with chemotherapy did not cluster together in a distinct group but rather were dispersed among the existing four clusters. Samples cc_29 and cc_76 clustered with the malignant adenocarcinomas, while samples cc_36 and cc_9 clustered with the benign adenomas. Sample cc_94 clustered with the borderline tumours. In an initial effort to test the possible clinical significance of the differential clustering of samples obtained from patients pre-treated with chemotherapy, we examined the post-operative history of these patients. One commonly used indicator of recurrence is the level of Cancer Antigen-125 (CA-125) in the blood [ 12 , 13 ]. Although post-operative CA-125 levels were initially lowered to a significant extent in all of the patients pre-treated with chemotherapy, the levels remained consistently low in only those patients (cc_36, cc_9) whose microarray profiles clustered with the benign adenomas (Figure 2 ). The remaining patients displayed periodic recurrence requiring additional chemotherapy. Figure 2 CA-125 levels of patients receiving chemotherapy prior to tissue collection. CA-125 levels of patients with cancer-like profiles (a) and adenoma-like profiles (b) were normalized to the earliest post-surgery reading. CA-125 level for patients 76 and 29, two patients receiving chemotherapy prior to tissue collection, spike dramatically at about 700 days post surgery. CA-125 levels for patients 9 and 36 remained low through 700 days past surgery. Significant differences in gene expression are associated with different ovarian tumour types To identify genes whose differential expression correlate with malignant potential, we performed a statistical analysis comparing the expression profiles of the three tumour types examined in this study (benign adenoma, low malignant potential borderline adenocarcinoma, and malignant adenocarcinoma). Malignant adenocarcinoma sample c_79 was excluded from this analysis since both the SOM and UHC classification methods identified this sample as an outlier of the malignant adenocarcinoma group (see above). The F statistic was used to test equality of group means [ 14 ]. Genes whose group means were identified as significantly different (p ≤ 0.001, 299 genes) in the ANOVA analysis were further analyzed using multiple comparison methods to determine which means differ from each other. The differences between group means for all pairwise combinations of groups were calculated and compared to the least significant difference. Genes were declared differentially expressed if the pairwise difference between group means was greater than the least significant difference. Probe sets duplicated between pairwise comparisons and probes sets with a fold change value below 2.0 were removed, leaving 163 unique genes differentially expressed between the tumour groups. The 15 differentially expressed genes with highest statistical significance are presented in Table 2 . The gene name, gene symbol, chromosomal location, functional classification, ANOVA rank and p-value of each of these 163 genes are attached as additional file 1 (complete list.txt). Table 2 Highest 15 statistically significant genes via ANOVA analysis, their fold change and p-values. Affy ID Gene Name Gene Symbol FC a:b FC a:c FC c:b ANOVA p-value 1651_at ubiquitin-conjugating enzyme E2C UBE2C 1.16(b) 4.35(c) 3.75(c) 1.2E-07 41583_at flap structure-specific endonuclease 1 FEN1 1.27(b) 3.86(c) 3.04(c) 1.9E-07 31888_s_at tumour suppressing subtransferable candidate 3 TSSC3 3.46(b) 8.15(c) 2.36(c) 2.7E-07 34715_at forkhead box M1 FOXM1 1.06(b) 2.66(c) 2.5(c) 7.5E-07 39109_at chromosome 20 open reading frame 1 C20orf1 1.29(b) 4.91(c) 3.80(c) 2.9E-06 37985_at lamin B1 LMNB1 1.19(b) 3.19(c) 2.69(c) 2.9E-06 41451_s_at SAR1 protein SAR1 1.07(b) 2.27(c) 2.13(c) 3.2E-06 37015_at aldehyde dehydrogenase 1 family, member A1 ALDH1A1 1.86(a) 10.76(a) 5.79(b) 3.3E-06 527_at centromere protein A, 17kDa CENPA 1.08(b) 3.47(c) 3.2(c) 3.9E-06 40619_at ubiquitin carrier protein E2-EPF 1.51(b) 2.74(c) 1.81(c) 4.6E-06 32332_at isocitrate dehydrogenase 2 (NADP+), mitochondrial IDH2 1.28(b) 4.36(c) 3.40(c) 5.4E-06 1058_at WAS protein family, member 3 WASF3 2.03(a) 2.56(a) 1.27(b) 5.7E-06 1943_at cyclin A2 CCNA2 1.05(a) 2.07(c) 2.17(c) 6.4E-06 2039_s_at FYN oncogene related to SRC, FGR, YES FYN 1.05(b) 3.11(a) 2.97(b) 6.4E-06 1868_g_at CASP8 and FADD-like apoptosis regulator CFLAR 0.97(b) 1.78(c) 1.83(c) 7.7E-06 Hierarchical clustering was performed to visualise gene expression across tumour types for each of these 163 genes. All 12 tumours were correctly assigned as shown by the dendogram above the gene expression colour plot (Figure 3a ). Several features within the gene expression colour plot are worthy of note (Figure 3b,3c,3d,3e,3f ). Thirteen genes (Figure 3b ) showed high expression in both adenoma and borderline. For forty genes expression levels in borderline tumours was intermediate between adenoma and cancer (Figure 3c ). Eight genes were highly expressed in either adenoma (3 genes, Figure 3c ) or borderline (5 genes, Figure 3d ). And finally, thirteen genes showed high expression in both cancer and borderline (Figure 3e ). Figure 3 Patterns of differential expression for the 163 genes of highest statistical significance. The 300 probe sets with the lowest p-values in the ANOVA analysis were filtered for duplicate genes and fold change <2.0. The remaining 163 genes were subjected to hierarchical clustering to reveal correlated expression (a). Thirteen genes showed high expression in both benign adenomas and borderline tumours (b). Borderline tumours showed intermediate levels of expression for forty genes (c). Three genes were high only in benign adenoma (d). Five genes showed high expression in borderline tumours only (e). And 13 genes were high in both malignant adenocarcinomas and borderline tumours (f). To independently test the validity of the differential expression patterns determined by microarray, we measured the expression patterns of 3 representative genes in 6 tissue samples using quantitative real time RT-PCR [ 15 ]. Genes were selected from the microarray data set to represent a spectrum of statistical significance (Table 3 ). In all cases, the results of the quantitative RT-PCR analyses confirmed the differences detected in the microarray studies (Figure 4 ). Table 3 Genes expression changes verified with quantitative RT-PCR. Gene Name Gene Symbol ANOVA rank p-value ubiquitin-conjugating enzyme E2C UBE2C 1 1.18E-7 cadherin 2, type 1, N-cadherin CDH2 177 0.00042 oviductal glycoprotein 1, 120 kDa OVGP1 739 0.0058 Figure 4 RT-PCR validation of microarray results. OVGP1 expression (a), CDH2 expression (b) and UBE2C expression as measured by RT-PCR (◆) and microarray (■). Patterns of change in expression were the same for each method. Functionally related genes display correlated changes in expression between benign malignant tumours Two expression subgroups were evident in the list of 163 differentially expressed genes (Figure 3a ): genes with low expression in cancer (first 61 genes of colour plot) and genes with high expression in cancer (last 102 genes of colour plot). To examine the possibility that these subgroups also correlate with differential gene function, we applied two functional profiling programs, EASE [ 16 ] and Onto Express [ 17 ]. Searching the gene ontology assignments for all genes in a list, these programs identify and assign statistical significance to the over-represented gene functional categories identifying common biological processes, molecular functions, cellular and chromosomal locations shared by genes in a list. Functional profiling revealed that the expression subgroups exhibited distinctly different gene functions (Figure 5 ). Genes in the expression subgroup with high expression in cancer were intracellular whereas the genes in the low expression subgroup were extracellular. Genes whose gene products function during cell proliferation and DNA metabolism dominate the high expression subgroup. On the other hand, gene products involving insulin-like growth factor binding, regulation of cell growth, cell-cell adhesion, and calcium transport activity were associated with the low expression subgroup. Figure 5 Over-represented functional categories of differentially expressed genes. Differentially expressed genes with low expression in cancer were able to bind insulin-like growth factor (p < 6 × 10 -6 ) or functioned in cell adhesion (p < 0.012) and calcium channel activity (p < 0.02). Genes with high expression in cancer functioned in nuclear division (p < 8 × 10 -8 ), mitosis (p < 5 × 10 -5 ), and DNA metabolism (p < 3 × 10 -8 ). Discussion Microarray profiles of ovarian tumours are of potential diagnostic and prognostic significance Gene expression profiling via microarray technology has previously been shown to be an effective tool for the objective classification of established tumour types [ 18 , 19 ] and in some instances, for the identification of previously unrecognized tumour sub-types [ 20 ]. Applied to ovarian cancer, gene expression profiling has aided in distinguishing clear cell carcinomas [ 8 , 9 ], characterizing advanced stage ovarian cancer [ 5 , 6 ], and identifying genes differentially expressed between normal and cancerous ovarian tissue [ 4 , 7 , 10 ]. The experiments presented here were designed to elucidate gene expression changes in ovarian tumours of differing malignant potential. In many instances, genes that we identified as differentially expressed across malignant potential were previously determined to be differentially expressed between normal and cancerous ovarian tissue including ERBB3 [ 10 ], ubiquitin carrier protein[ 10 ], and E-cadherin [ 4 ]. We also correctly classified 92% of tumours from patients who did not receive chemotherapy prior to surgery into their proper histopathological group. These results are consistent with earlier findings and indicate that gene expression profiling can effectively distinguish between malignant and benign ovarian tumours. One particularly promising result emerging from our study is that expression profiling may be useful in predicting recurrence in patients treated with chemotherapy prior to surgery. We find that the microarray patterns of ovarian adenocarcinomas obtained from patients treated with chemotherapy prior to surgery clustered either with the benign tumours or with the malignant adenocarcinomas. Serum CA-125 levels indicate that patients whose samples clustered with the benign tumours have remained disease free for more than 3 years after surgery while those patients whose samples clustered with the malignant tumours recurred within 2 years of the initial treatment. Clearly, the testing of additional patient samples will be needed before definitive conclusions can be drawn. However, the preliminary results are consistent with the hypothesis that gene expression profiles of samples removed on the day of surgery may predict recurrence and would therefore be an indicator of the long-term effectiveness of chemotherapy administered to patients prior to surgery. Expression profiles indicate that borderline tumours are not a distinct disease Our microarray data are, in general, most consistent with the hypothesis that borderline ovarian tumours represent an intermediate stage between the benign and malignant tumours. Borderline tumours of the ovary display many but not all characteristics of malignancy including nuclear atypia and increased mitotic count, usually in the absence of stromal invasion [ 21 - 24 ]. Whether the borderline tumour is a precursor to the fully malignant ovarian carcinoma or a disease distinct from invasive carcinomas is a topic that has been debated since the International Federation of Gynaecologic Oncology added the borderline tumour to the classification of ovarian tumours in 1972. Distinct disease states are expected to show discrete gene expression patterns when analysed by microarray [ 3 ]. Our analysis identified only 5 genes (Figure 3e ) with increased expression distinctly correlated with borderline tumours. On the other hand, for 40 of the 163 genes displaying a significant change in expression between benign and malignant ovarian tumours, borderline tumours display an intermediate expression level (Figure 3c ). In all other cases, (118 genes) borderline expression mimicked either the benign adenoma (102 genes) or malignant adenocarcinoma (16) tumours. Thus, for these genes, borderline tumours appear to be a transitional state between the benign and malignant state. The five genes identified as characteristic of borderline tumours (Figure 3e ) may constitute a reliable marker of borderline tumours. Interestingly, two of these genes, AGR2 and NPTX2, are physically linked to one another, mapping to p21.3 on chromosome 7. Many genes displaying altered patterns of expression between benign and malignant ovarian tumours are genetically linked Genes physically linked to one another shared changes in gene expression between tumour types. For those cases where linked genes displayed a significant reduction in expression in malignant vs. benign tumours (Table 4 ), at least three explanations are possible. Perhaps the most likely explanation is that the change is due to a small deletion in a chromosomal region encompassing the affected alleles. Such deletional events are believed to be at the basis of the "loss of allele" (LOA) phenomenon, which is known to be a relatively common event in tumour development [ 25 - 27 ]. Another possibility is that these co-ordinated reductions in gene expression are due to regional changes in chromatin structure resulting in the reduced access of transcription factors to genes. Such epigenetic changes are typically associated with the hypermethylation of so-called "CpG islands" in or around genes [ 28 - 30 ]. Indeed, it has been well documented that the silencing of many tumour suppresser genes and genes involved in DNA repair and apoptosis in cancer cells is the consequence of DNA hypermethylation [ 31 , 32 ]. The third possibility is that the coordinated reductions are the result of completely independent mutational events. However, the probability that such independent events would repeatedly occur at linked loci seems low. Table 4 Co-ordinately expressed genes sharing chromosomal location Gene Symbol Location Expression in cancer Function KIF2C 1p34.1 Up Nuclear division/mitosis CDC20 1p34.1 Up Regulation of cell growth PMSB2 1p34.2 Up Protein Catabolism UBE2C 20q13.12 Up Nuclear division/mitosis STK6 20q13.2-q13 Up Signal Transduction RGS19 20q13.3 Up Nuclear division/mitosis PDGFRA 4q11-q13 Down Regulation of cell growth IGFBP7 4q12 Down Regulation of cell growth HNRPDL 4q13-21 Down RNA binding FYN 6q21 Down Calcium ion transport LAMA2 6q22-q23 Down Cell adhesion CTGF 6q23.1 Down Cell adhesion FOXM1 12p13 Up Transcriptional regulation CCND2 12p13 Down Nuclear division/mitosis ERBB3 12p13 Up Signal Transduction We also observed a co-ordinated increase in gene expression of physically linked genes in the malignant samples in several cases (Table 4 ). These changes may have been due to regional duplication or amplification events. Examples of such events have been previously documented in cancer cells [ 33 - 35 ]. It is also possible that at least some of these co-ordinated increases in gene expression are the consequence of regional hypomethylation events resulting in a more open chromatin configuration and a consequent increase in transcription factor accessibility. Genes located in proximity to transposable element sequences may be more prone to such epigenetic events [ 36 ]. In a few instances genes that were physically linked displayed opposing changes in gene expression between benign and malignant tumours (Table 4 ). It is possible that these disparate changes were due to independent mutational events or, perhaps more likely, to a regional relaxation of chromatin structure that permitted increased access of both positive and negative transcription factors. Our finding that a number of the genes displaying a significant difference in expression among malignant and non-malignant tumours indicates that some caution must be taken in the functional interpretation of microarray results. For example, significant changes in the expression of only one gene in a physically linked group may be of functional significance although correlated changes in gene expression may result from a regional effect. Malignant ovarian tumours display expression profiles consistent with previously established features of cancer cells Two common features of malignant cancer cells are increased cell proliferation and loss of cell adhesion [ 37 ]. Consistent with these general features, we found that the majority of differentially expressed genes with high expression in the malignant tumours belonged to functional categories associated with DNA metabolism and cell proliferation. We also report here that insulin-like growth factor binding, cell adhesion and calcium ion transport were gene functional categories over-represented among the genes significantly under-expressed in ovarian cancer. The IGF system is a complex network of molecules involved in the normal growth and development of many cell types [ 38 ]. Disregulation of the IGF system through over-stimulation of the IGF1 receptor (IGF1R) has been implicated in tumour development and maintenance of the transformed phenotype [ 39 , 40 ]. The functional consequences of IGF1R over-stimulation include increased cell proliferation, cell survival and regulation of cell adhesion. The six specific IGF binding proteins (IGFBP-1 through -6) bind IGF in the serum and extracellular matrix, thereby reducing the bioavailability of IGF1 for receptor binding, as well as downstream signalling. Recently, elevated serum levels of IGFBP-2 at diagnosis were correlated with the likelihood of relapse, confirming the prognostic value of serum IGFBP-2 in choosing aggressive treatments for these patients[ 41 ]. Measuring serum levels of IGFBP-3 and IGF1 of healthy women proved useful in predicting a woman's risk of ovarian cancer [ 42 ]. Our analysis demonstrated significantly lower expression of IGFBP-4, -5, and -7 in the malignant adenocarcinomas than in the benign adenomas or borderline tumours (Figure 6b ). IGFBP-2 and -3 were highly expressed but not differentially expressed across the tumour types. Furthermore, no IGF binding proteins appeared in the list of genes significantly up-regulated in cancer tissue. These findings suggest that loss of expression of IGFBPs in adenocarcinomas increases IGF signalling and its functional consequences, processes clearly associated with the clinical phenotypes of ovarian adenocarcinomas. Figure 6 Microarray expression of cadherins and insulin-like growth factor system genes. E-cadherin expression contributes about equally to the cadherin distribution in benign adenomas but is the dominant component of the cadherin distribution in malignant adenocarcinomas (a). Insulin-like growth factor system components show lower expression in the adenocarcinomas relative to benign adenomas and borderline tumours (b). P-values associated with differential expression as analysed by ANOVA analysis are shown above each genes column graph. Over-expression of certain members of the IGF system increased sensitivity to IGF1 signaling in breast cancer cells [ 43 ] leading to increased cell proliferation. Insulin receptor substrate 1 (IRS1) is one member of the IGF system whose over-expression potentiated the effects of IGF1. Interestingly, IRS1 was significantly up-regulated in the benign and LMP tumours of our study (Figure 2 ). Considering the documented ability of the IGFBP's to reduce bioavailability of IGF1, increased expression of IGFBP's would be an appropriate cellular response to increased expression of IRS1. Loss of cell adhesion molecules (CAM) is one mechanism proposed to induce the tissue invasion and metastatic capabilities acquired by cells during tumourigenesis[ 37 ]. Intra-abdominal spread of ovarian cancer via peritoneal implants is a hallmark of advanced stage ovarian cancer and can be linked to loss of cell-cell adhesion [ 44 ]. Our findings support the theory that loss of CAM in ovarian cancer is instrumental in cancer progression. Cell-cell adhesion is often mediated through the cadherins, a family of transmembrane glycoproteins that require calcium to perform their adhesive functions. Well-documented changes in cadherin subtype expression correlate with the progression of breast and prostate cancer [ 45 ]. Recently, differences in the profile of cadherin subtypes expressed in normal and cancerous ovarian tissue were also shown to correlate with disease progression [ 46 ]. Support for cadherin switching in ovarian tumours is evident in our microarray data. Expression of N-cadherin (N-cad) and cadherin-11 (CDH11), the dominant subtypes in normal ovarian surface epithelium, were significantly higher in the benign and LMP tumours of our study than in the adenocarcinomas (Figure 4a ). The intensity of change in expression between the benign adenomas and malignant adenocarcinomas for N-cad and CDH11 were 3.9 and 7.8 fold respectively, and both genes appeared in the list of top 163 differentially expressed genes. The LMP tumours in our study expressed N-cad and CDH11 at levels intermediate to adenomas and adenocarcinomas, suggesting an integral role for these cadherins in transformation to a malignant phenotype. Expression of E-cadherin, a major subtype seen in adenocarcinomas, increased approximately 2 fold from a benign tumour to either LMP or the adenocarcinomas (Figure 6a ). This data documents the switch from a normal-like distribution of N-cad and CDH11 in the benign and LMP tumours to a cancerous profile dominated by E-cad expression. Since cadherins are calcium-dependent cell adhesion molecules [ 44 ]and increased dietary intake of calcium correlates with a reduced risk of ovarian cancer [ 47 ], it is also interesting that calcium transport and calcium channel activity are gene functions that we found correlated with genes under-expresses in the adenocarcinomas. Thus it is also possible that altered functionality of the cadherins through changes in calcium availability, a parameter not measurable with microarray, may be involved in increasing a tumours' malignant potential. Conclusions Our findings indicate that gene expression profiling can reliably distinguish between benign and malignant ovarian tumours. Expression profiles of samples from patients pre-treated with chemotherapy may be useful in predicting disease free survival and the likelihood of recurrence. Genes displaying co-ordinated changes in gene expression were often genetically linked suggesting that changes in expression for these genes are the consequence of regional duplications, deletions or epigenetic changes. Loss of expression of IGF binding proteins as well as specific cell adhesion molecules may be a significant mechanism of disease progression in ovarian cancer. A significant portion of the differentially expressed genes exhibited expression levels in borderline samples intermediate between benign adenomas and malignant adenocarcinomas, suggesting the borderline tumours are a transitional state between benign and malignant tumours. Methods Tumour Samples and RNA Isolation A set of 18 primary ovarian tumours was obtained from the Ovarian Cancer Institute. This set of tumours was comprised of 4 benign cystadenofibromas, 4 carcinomas of low malignant potential (borderline carcinomas), 5 adenocarcinomas, and 5 adenocarcinomas from patients who received chemotherapy prior to surgery. This study was approved by the Institutional Review Board of the University of Georgia and of Northside Hospital (Atlanta), from which the samples were obtained Tissue was collected at the time of initial surgery and preserved in RNA Later (Ambion) within one minute of collection. For RNA isolation, each tissue (50 ± 25 mg) was homogenized on ice in 1.5 ml Trizol (Molecular Research Corporation) with a polytron homogenizer for about 30 seconds. RNA was isolated from the crude homogenate according to the manufacturer's protocols (Trizol, Molecular Research Corporation) with the following specifics. Linear polyacrylamide (5 μl) was added prior to homogenization to aid in RNA precipitation. Total RNA was further purified over an RNEasy (Qiagen) column using the manufacturer's cleanup protocol. Microarray Hybridization Biotinylated target cRNA was generated according to the Affymetrix Technical Manual. In brief, 5–10 μg total RNA was converted to double stranded cDNA using Supercript II (Invitrogen). The cDNA was cleaned by phenol/chloroform extraction and ethanol precipitation. In vitro transcription of the cDNA with the High Yield RNA Transcript Labeling Kit (Enzo) yielded 50–100 μg of biotin labeled cRNA target. The cRNA was fragmented in a metal catalyzed acid hydrolysis to a length of 20–200 bp (by electrophoresis) and the fragmented cRNA was hybridized to the Affymetrix array (U95Av2) for 16 hours at 45C. Hybridized arrays were washed, stained and scanned according to the Affymetrix technical manual. Microarray Data Handling and Manipulation Signal values were generated in two ways. Affymetrix signal values were generated from the .CEL file using the Affymetrix software MAS 5.0. The overall intensity of each array was scaled to an average intensity of 500. These normalized signal values were exported to Excel (Microsoft) for further analysis labeled the Affy-data set. Robust multi-array analysis (RMA) signal values were generated from the .CEL file using the espresso wrapper in the Affy library of the Bioconductor package in the R-statistical environment. The parameters of PM correction, background correction, normalization, and summary method were set to PM only, RMA, quantile, and median polish, respectively. The normalized signal values were exported to Excel for further analysis and will be referred to in this paper as the RMA-data set. For each data set, Affy-data set and RMA-data set, Pearson correlation coefficients were calculated (Microsoft, Excel) for a_97 vs. all other arrays. Higher correlation and lower standard deviation from the mean within groups was seen with the RMA data set, suggesting higher quality data. Clustering Raw data output from the Affymetrics MicroArray reader is transformed into expression level values using the RMA method [ 48 , 49 ]of the "affy" package in the Bioconductor suite of the R statistical environment, and a text output file generated. This text file is then transformed into a *.gct file for input into the GeneCluster program. GeneCluster (Whitehead Insitute, ) was used to cluster the dataset on both samples and genes. Except where described below, default parameters were used. The SOM feature of GeneCluster was employed, and various values were explored for the "Cluster Range" and "Iterations" parameters. The 'Cluster Range' parameter sets the geometry of the clusterings that will be performed on the data. For instance, if 2–3 is entered, two cluster sets are produced. One has two clusters and the other three clusters. When entering a number, any set of factors of that number will create a clustering. If 9 is entered, a linear set of 9 clusters and a 3 × 3 matrix of clusters are produced. Marker analysis was also performed in GeneCluster, again using the default parameters. Quantitative RT-PCR Total RNA (2 μg) from ovarian tissue was converted to cDNA using Superscript III (Invitrogen) primed with random hexamers under conditions described by the supplier. cDNA from this reaction was used directly in the Quantitative RT-PCR analysis. TaqMan probes and gene specific primers for three genes (RPL-29, UBE2C, OVGP1, and CDH2) were obtained from Applied Biosystems' Assay on Demand. The mRNA levels of the three genes were measured in 6 ovarian tumours and one normal ovary on the ABI Prism 7700 Sequence Detection System. PCR was performed using the TaqMan Universal PCR MasterMix (Applied Biosystems), according to the manufacturer's protocols with standard PCR cycling steps. Using RPL29 as a housekeeping gene and the normal human ovary RNA as a reference sample, the expression levels of UBE2C, OVGP1 and CDH2 were calculated according to the 2 -ΔΔCt method[ 15 ]. The Ct values of triplicate RT-PCR reactions were averaged for each gene in each cDNA sample. For each tissue sample assayed, the average Ct value for the gene of interest (UBE2C, CDH2 and OVGP1) was subtracted from the average Ct value of the housekeeping gene (RPL29) to obtain the ΔCt value. The ΔCt value of the reference sample was subtracted from that of the tumours to obtain the ΔΔCt value. Data Filtering and Statistical Analysis The RMA normalised data set was analysed for probe sets likely to be absent in all samples. Probe sets whose maximum RMA normalised value across all samples was less than 5.2 were removed from further analysis. Analysing the remaining 10,520 probe sets, we applied an analysis of variance (ANOVA) to test the hypothesis that the mean expression values for all groups (adenoma, borderline and cancer) are equal. For each gene, the within group and between group variation was calculated and used to generate the F statistic and subsequent p-values [ 14 ]. Adjusted p-values were also calculated using Holm's method. The ranking of genes in order of significance was exactly the same for the un-adjusted and adjusted methods. However, the adjusted p-values were 1000 fold higher than the unadjusted p-values and only the top 50 genes were considered significant (p < 0.05). Since RT-PCR confirmed differential expression down to the 739 th statistically significant gene (see results section), we continued the analysis on the top 300 statistically significant genes. Genes whose groups means were identified as significantly different (p ≤ 0.001, 299 genes) in the unadjusted ANOVA were further analysed using Fisher's Least Significant Difference multiple comparison method. The differences between group means for all pairwise combinations of groups were calculated and compared to the least significant difference. Genes were declared differentially expressed if the pairwise difference between group means was greater than the least significant difference. Probe sets duplicated between pairwise comparisons and probes sets with a fold change value below 2.0 were removed, leaving 163 unique genes differentially expressed. Functional Profiling Genes found to be differentially expressed in the statistical analysis were divided into two lists: genes overexpressed in cancer, and genes underexpressed in cancer. Each list was analyzed for over-represented functional categories based on molecular function, biological process, cellular component and chromosomal location using two different freeware programs: EASE [ 16 ]and OntoExpress [ 17 ]. Given a list of genes, EASE forms subgroups of genes based on the functional categories assigned to each gene. EASE assigns a significance level to the functional category based on the probability of seeing the number of subgroup genes within a category given the frequency of genes from that category appearing on the microarray. The 'EASE score' is the upper bound of the distribution of Jacknife Fisher exact probabilities. Onto Express identifies overrepresented gene functional categories in a manner similar to EASE and was used to verify results obtained from EASE. All information on chromosomal location was obtained from Onto Express since EASE does provide information on chromosomal location. Authors' contributions SW performed microarray, basic statistical analysis and hierarchical clustering. SP performed SOM clustering and marker analysis. SD contributed to the statistical analysis. BB collected tissue and serum. EK directed SW and SP. JM contributed to the linkage and CA-125 analyses and directed SW. Supplementary Material Additional File 1 The file complete_list.xls contains the gene name and chromosomal location for the 163 genes determined to be differentially expressed in this study. Click here for file | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524500.xml |
516029 | In situ transduction of stromal cells and thymocytes upon intrathymic injection of lentiviral vectors | Background The thymus is the primary site for T-cell development and induction of self-tolerance. Previous approaches towards manipulation of T-cell differentiation have used intrathymic injection of antigens, as proteins, cells or adenoviruses, leading to transient expression of the foreign protein. Lentiviral vectors, due to their unique ability to integrate into the genome of quiescent cells, may be best suited for long-term expression of a transgene in the thymus. Results Young adult mice were injected in the thymus with lentiviral vectors expressing eGFP or the hemaglutinin of the Influenza virus under the control of the ubiquitous phospho glycerate kinase promoter. Thymi were examined 5 to 90 days thereafter directly under a UV-light microscope and by flow cytometry. Intrathymic injection of lentiviral vectors predominantly results in infection of stromal cells that could be detected for at least 3 months. Importantly, hemaglutinin expression by thymic stromal cells mediated negative selection of thymocytes expressing the cognate T-cell receptor. In addition and despite the low multiplicity of infection, transduced thymocytes were also detected, even 30 days after injection. Conclusions Our results demonstrate that intrathymic delivery of a lentiviral vector is an efficient means for stable expression of a foreign gene in the thymus. This new method of gene delivery may prove useful for induction of tolerance to a specific antigen and for gene therapy of severe combined immunodeficiencies. | Background The thymus is a bilobate organ derived from embryonic endoderm and mesoderm differentiation and is located just above the heart (reviewed in [ 1 ]). It is the primary organ for maturation of T cells. This process involves the interaction between developing thymocytes and thymic stromal cells. Thymic stromal cells which forms the thymic architecture, have been classified according to their anatomical localization. They encompass a very diverse array of cell types, including cortical and medullar epithelial cells, fibroblasts, macrophages and dendritic cells (reviewed in [ 2 ]). Stromal cells control the differentiation of haematopoietic precursors derived from the liver or the bone marrow into T lymphocytes: T-cell differentiation is defined by the acquisition of maturation markers such as CD4, CD8 and the T-cell receptor complex (TCR), which conditions the reactivity of immature thymocytes with thymic stromal cells. The early thymocyte progenitors entering the thymus do not express T-cell-specific molecules, such as CD3, the alpha or beta-chain of the TCR, or the CD4 and CD8 molecules. These CD4 - CD8 - cells, referred to as double-negative (DN) cells, then become CD4 + CD8 + , the so-called double-positive (DP) stage, and then progressively acquire TCR molecules. The final maturation of T-cells involves the selective loss of either the CD4 or the CD8 molecules to generate fully mature single-positive (SP) cells with cytotoxic/suppressor or helper/regulator function, respectively. During this process, the TCR-mediated positive and negative selection of T cells ensures the selection of a diverse TCR repertoire able to react with foreign peptide presented by autologous major histocompatibility complex (MHC) molecules, but tolerant to self-antigens. This property renders the thymus an attractive site for manipulation of T-cell tolerance. To date, results on tolerance induction via direct manipulation of the thymus have been scarce (reviewed in [ 3 ]). However, previous studies using intrathymic (IT) injection of pancreatic islet cells [ 4 ], soluble antigens [ 5 ] or adenoviral vectors [ 6 , 7 ] have shown that induction of tolerance to foreign antigens in non-immunosuppressed animals is feasible. Since the production and maturation of thymocytes may be a life-long process, a major drawback to the utilisation of soluble antigens or adenoviruses is their short-term expression in the thymus [ 8 ]. Indeed, modulation of the selection process should stop upon the disappearance of the antigen, which might be a problem for long-term tolerance induction. Due to their ability to infect resting cells and to stably integrate into the genome, lentiviral vectors represent powerful new tools for long-term expression of a given transgene in vivo [ 9 ]. Lentiviral vectors have been used successfully in vivo to infect hepatocytes and muscle cells [ 10 ], antigen-presenting cells [ 11 , 12 ], as well as cells of the central nervous system [ 13 ]. We reasoned that lentiviral vectors might be better suited than adenoviral vectors for long-term IT expression of a foreign gene. We therefore investigated the pattern of infection of a ubiquitous lentiviral vector after IT injection. We report herein that thymic stromal cells are massively and persistently infected. Developing thymocytes exhibit a significant but low level of infection. Moreover, we show that IT injection of a lentiviral vector encoding the cognate antigen in TCR-transgenic (Tg) mice leads to negative selection of developing thymocytes. Results Intrathymic injection of lentiviral vectors results in the efficient and persistent infection of thymic stromal cells We used a concentrated viral stock of the LvPGK-GFP vector (2.10 9 TU 143B /ml) to inject between 7 × 10 7 to 1.2 × 10 8 infectious units in the thymus of normal C57Bl/6 mice. Infected cells could readily be detected at day 5 post-injection by direct examination of the thymi under a UV microscope (figure 1 ). Of note, transduced cells could still be observed at 1 (figure 1 ) and 3 months (data not shown) post injection. Most of the transduced cells had a fibroblastic morphology. To monitor a possible passage of the vector through the bloodstream, we checked for the presence of transduced cells in the liver, which is the primary target organ after intravenous injection of lentiviral vectors [ 14 ]. We indeed observed numerous eGFP + cells in the liver (figure 1 ), suggesting a significant leak into the circulation upon IT injection of up to 30 μl of the vector. Nevertheless, our results demonstrate efficient and persistent infection of thymic stromal cells upon IT injection of a lentiviral vector. Figure 1 In vivo expression of eGFP after intrathymic injection of the LvPGK-GFP vector. D5: day 5 post-injection localisation of transduced cells around the injection site (arrow) under visible and UV-lights (upper picture). Fibroblast-shaped cells are predominantly transduced (UV-light only) (lower panel). D30: day 30 post-injection expression of eGFP in the thymus (visible + UV-light) (upper panel) and in the liver (UV-light only) (lower panel). CTRL: Thymus (upper panel) and liver (lower panel) pictures from control mice injected IT with PBS examined for background fluorescence under visible and UV-lights. Magnifications are indicated in the lower right corner of each picture. Induction of negative selection after intrathymic injection of lentiviral vectors We next wanted to investigate whether thymic stromal cells infected with a lentiviral vector would be able to mediate negative selection of developing thymocytes. We thus injected a lentiviral vector encoding the HA of the Influenza virus, or eGFP as a control, into the thymus of SFE-Tg mice expressing a TCR specific for a HA protein peptide and for which a clonotypic antibody recognizing the transgenic TCR (clone 6.5) is available [ 15 ]. Six days after injection, we analyzed the thymocytes by flow cytometry after thymi dilacerations. The frequency of 6.5 + cells within CD4SP and CD8SP thymocytes is shown in figure 2A for a representative experiment. Intrathymic injection of the LvPGK-HA vector resulted in a diminution in the frequencies of 6.5 + cells within CD4SP by a factor of 2 and by a factor of 5 in CD8SP cells with an almost complete disappearance of 6.5 hi cells in the latter subset. Of note is that the intensity of TCR transgenic expression was reduced in CD4SP cells (figure 2A ). Overall, we observed a 3.5-fold decrease in the total numbers of thymocytes expressing the specific TCR in mice injected with the LvPGK-HA vector compared to the LvPGK-GFP vector (figure 2B ). Our results altogether demonstrate that within a week after intra thymic injection of the LvPGK-HA vector, infected thymic stromal cells efficiently mediated negative selection of HA-specific thymocytes. Figure 2 Negative selection of developing thymocytes (A) TCR transgenic expression within thymic CD8SP (white) and CD4SP cells (grey) identified by the anti-clonotypic monoclonal antibody 6.5 in SFE-Tg mice six days after IT injection of 40 to 60 ng p24 of the LvPGK-GFP lentiviral vector (n = 2) or of 3.5 to 6 ng p24 of the LvPGK-HA vector (n = 3). Shown are representative profile of two independent experiments. Numbers indicate the frequency of 6.5 + cells (B) Absolute counts of HA-specific thymocytes six days after intra thymic injection of LvPGK-GFP or LvPGK-HA lentiviral vectors. These figures were obtained based on the percentages of total 6.5 + thymocytes determined by flow cytometry as shown in (A). Statistical analysis was performed using Student's t-test. Intrathymic injection of lentiviral vectors results in low level infection of immature thymocytes We next investigated whether developing T-cells would be infected upon IT injection of the lentiviral vector. Five days post-injection of the LvPGK-GFP vector into the thymus of a normal mouse, very few eGFP + cells could be detected by flow cytometry within the thymocytes obtained from dilacerated thymi (figure 3 ). Most of these cells were CD3 - cells, and belonged to the DN and DP subsets, showing that infected cells were mostly immature. Interestingly, at day 30 post-injection, we observed more mature eGFP + cells that expressed CD3 and that belonged to the subsets of CD4SP and CD8SP for more than half of them. This result indicates that infection per se did not interfere with the normal process of T-cell development. Moreover, we observed a similar repartition of CD4/CD8-expressing cells in non-infected eGFP - cells (data not shown). Collectively, these results show that immature thymocytes can be infected by in situ lentiviral infection. However, we were unable to clearly detect infected T-cells in the spleen of injected mice, likely due to their small representation within the pool of mature lymphocytes in the absence of a selective advantage for the transduced thymocytes. Figure 3 eGFP expression in developing thymocytes. Total thymocytes were stained with anti-CD4, anti-CD8 and anti-CD3 monoclonal antibodies. Upper panels: saline-injected control mice (CTRL) and LvPGK-GFP-injected mice at two different time points after injection (D5 and D30) are shown. Lower panels: The profile of CD4/CD8 expression is shown within gated eGFP + cells. Discussion We report herein that IT injection of a lentiviral vector results in the predominant infection of thymic stromal cells, and to a low level infection of thymocyte progenitors. Significant infection of liver cells was also detected. This observation is reminiscent of what was observed by DeMatteo et al. with adenoviral vectors [ 7 ]. Together with the fact that liver cells are main targets of IV-injected lentiviral or adenoviral vectors, this suggest that a significant leak into the circulation does occur upon IT injection of viral vectors. Our in situ analysis suggests that thymic epithelial cells represent the vast majority of infected cells, and studies are underway to more precisely define the cells targeted by IT lentiviral injection. Whatever the proportion of cortical, medullar epithelial cells, or thymic dendritic cells that are transduced, we show here that this results in an antigen presentation that efficiently mediates negative selection of specific thymocytes. This is not due to the injection of a "crude" preparation of viral supernatant that could have non-specifically affected T cell differentiation. Indeed, we injected 10 times lower amounts of p24 from the LvPGK-HA vector than of the LvPGK-GFP vectors, suggesting that negative selection of HA-specific thymocytes was a direct effect of HA expression by thymic stromal cells. This is further supported by the analysis of the frequencies of 6.5 + thymocytes which shows deletion of 6.5 hi cells within CD8SP cells, an MHC class-II restricted population in these TCR-transgenic mice [ 15 ]. Down modulation of the transgenic TCR and deletion was observed within CD4SP cells. This is reminiscent of the results obtained recently by Trani et al. which showed that intra thymic delivery of increasing dose of the HA peptide in SFE TCR-Tg mice resulted in the down regulation of the transgenic TCR [ 16 ]. Therefore, deletion and/or receptor down regulation may act in concert in the negative selection of HA-specific CD4SP cells in SFE transgenic mice. A very low infection of developing thymocytes was detected. This is not surprising as (i) the multiplicity of infection (ratio of number of infectious units over number of total cells in the thymus) was estimated to be lower than 0.4 and (ii) lentiviral transduction of murine T cells is far less efficient than of human T cells [ 17 ]. Since the actual volume that can be injected in a mouse thymus is however limited, we used the highest MOI achievable with our concentrated vectors. It would be interesting to assess if the use of vectors with higher infectious titres or of repeated injections would lead to a better transduction of thymocytes. It should be stressed though that at day 5 after injection, the infected cells represented immature thymocytes not expressing CD3 molecules. At later time points, infection was detected in more mature thymocytes. We believe that this result may have important implications for in vivo gene therapy of severe combined immunodeficiencies (SCID) affecting T-cell development (reviewed in [ 18 ]). Indeed, most of these diseases are due to monogenic mutations and concerns immature thymocytes, such as in the T - B + NK - deficiencies linked to the common cytokine receptor gamma-c [ 19 ], or to the ZAP-70 protein tyrosine kinase [ 20 ]. Our results open the possibility of correcting these developmental blocks through IT delivery of a lentiviral vector expressing a functional molecule. For this particular application, it would be important to avoid transgene expression in the thymic stroma. The use of our recently described T-cell specific lentiviral vector represent an attractive possibility towards this end [ 21 ]. Given the tremendous proliferative potential of T cells and the selective advantage that will be provided by the transgene, even a low number of transduced cells should result in a significant T cell reconstitution. This is best exemplified by a unique case of X-linked severe combined immunodeficiency in which a reverse mutation occurred in a single early T cell precursor. It was determined that at least 1,000 T cell clones with unique T cell receptor-beta sequences were generated from this precursor and that this diversity seems to be stable over time and provides protection from infections in vivo [ 22 ]. Furthermore, our preliminary results show that intra thymic delivery of the ZAP-70 gene by mean of a T-cell specific lentiviral vector in ZAP-70-deficient mice results in the restoration of T-cell development (submitted). The presently described in vivo approach may represent an alternative to gene therapy protocols using cumbersome haematopoietic stem cell manipulation ex vivo prior to their reinfusion in vivo . Conclusions Results presented herein may have important implications for the experimental and therapeutic manipulation of the immune system, and notably for tolerance induction and the correction of SCID. Methods Mice and intrathymic surgery C57Bl/6 mice were obtained from Charles River/IFFA Credo Laboratories (Les Oncins, France) at 6 weeks of age and were used at 8 to 10 weeks-old. SFE TCR-Tg mice [ 15 ] were bred in our own animal facility and were used at 6 to 10 weeks-old. Intrathymic surgery was performed after anesthetic treatment of animals with 40 mg/kg of Pentobarbital (Sanofi-Synthelabo, Gentilly, France). Mid-incision of the lower neck was performed to gain access to the trachea. Incision of the sternum was performed on the first two ribs and gently pulled aside to view the thymus. A single injection of 10 to 30 μl was performed using 0.3 ml Terumo insulin syringes (VWR, Fontenay-sous-bois, France). Lentiviral vector construction, production, concentration and quantification The plasmid encoding the lentiviral vector pRRLsin.PPT.hPGK.GFPpre (LvPGK-GFP) has been described elsewhere [ 23 ] (kindly provided by L. Naldini (University of Torino Medical School, Torino, Italy). To construct the plasmid encoding the hemaglutinin (HA) protein of the Influenza virus, BamHI and SalI restriction sites at the 5' and 3' ends, respectively, were added to the cDNA of the HA protein of Influenza virus (H1N1) in the pCIneoHA plasmid (provided by Genethon, Evry, France) by PCR using the Taq polymerase (Invitrogen, Cergy-Pontoise, France). Total PCR products were cloned into the TA vector (Invitrogen), checked for sequence integrity and digested with BamHI/SalI. The plasmid pRRLsin.PPT.hPGK.GFPpre was digested with BamHI/SalI (New England Biolabs, Beverly, Massachussets, USA) to remove eGFP. After ligation, the plasmid pRRLsin.PPT.hPGK.HApre, hereafter referred to as LvPGK-HA, was obtained. To produce lentiviral vectors, a total of 4.10 6 293T-cells were co-transfected with the transfer vector, the packaging and the envelope plasmids in 10-cm dishes using the calcium phosphate method as described [ 21 ] in DMEM supplemented with serum and antibiotics (Lifetechnologies, Gaithersburg, Maryland, USA). Lentiviral supernatants were collected at 18, 42 and 66 hrs post co-transfection in serum-free DMEM supplemented with antibiotics and L-glutamine, and concentrated by ultrafiltration using either the Ultrafree-15 or the Centricon Plus-80 filter devices according to the manufacturer instructions (Millipore, Bedford, Massachussets, USA). Briefly, supernatants were applied to the filter devices and spun at 2000 g for 20 min. at 20°C. Concentrated supernatants were aliquoted and kept at -80°C until use. Viral stocks of the LvPGK-GFP lentiviral vector were titered on 143B cells as previously described [ 21 ]. Viral stocks of the LvPGK-GFP and LvPGK-HA vectors were also quantified using a gag p24 ELISA (Zeptometrix, Buffalo, New York, USA). Microscopy and images treatment Whole thymus or liver were excised from injected mice and placed in PBS 1X in a 6-well plate. Pictures of the whole organ were acquired using a DP-11 numeric camera coupled with the CK-40 inverted microscope equipped with a mercury lamp (Olympus France S.A, Rungis, France). Images were processed using Adobe Photoshop (Adobe Systems Inc., San Jose, California, USA). Flow cytometry Thymi were dilacerated between two frosted slides in 1X PBS supplemented with 3% Fetal Calf Serum (Lifetechnologies). Cell suspensions were numerated and 10 6 cells were stained with the following monoclonal antibodies (Becton Dickinson Biosciences, le Pont de Claix, France): CD4-APC (allophycocyanin), CD8-CyCr (Cychrome) and either CD3 or pan beta-chain of the TCR-PE (phycoerytrin) or purified anti-clonotypic TCR for the HA peptide SFERFEIFPK presented by MHC class II I-E d (clone 6.5) ([ 15 ]) followed by biotinylated anti-rat IgG2b and streptavidin-FITC. Data were collected on a FACScalibur (BD Biosciences) and analysed with FlowJo software (TreeStar, Ashland, Oregon, USA). Abbreviations DN: double negative DP: double positive SP: single positive IT: intra thymic IV: intra venous HA: hemaglutinin MOI: multiplicity of infection SCID: severe combined immunodeficiencies Authors' contributions GM devised and realised the experiments, analysed the data and wrote the manuscript. DK conceptualised the study and edited the manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516029.xml |
314465 | Loss of Olfactory Receptor Genes Coincides with the Acquisition of Full Trichromatic Vision in Primates | Olfactory receptor (OR) genes constitute the molecular basis for the sense of smell and are encoded by the largest gene family in mammalian genomes. Previous studies suggested that the proportion of pseudogenes in the OR gene family is significantly larger in humans than in other apes and significantly larger in apes than in the mouse. To investigate the process of degeneration of the olfactory repertoire in primates, we estimated the proportion of OR pseudogenes in 19 primate species by surveying randomly chosen subsets of 100 OR genes from each species. We find that apes, Old World monkeys and one New World monkey, the howler monkey, have a significantly higher proportion of OR pseudogenes than do other New World monkeys or the lemur (a prosimian). Strikingly, the howler monkey is also the only New World monkey to possess full trichromatic vision, along with Old World monkeys and apes. Our findings suggest that the deterioration of the olfactory repertoire occurred concomitant with the acquisition of full trichromatic color vision in primates. | Introduction Olfactory receptor (OR) genes provide the basis for the sense of smell ( Buck and Axel 1991 ) and, with more than 1,000 genes, comprise the largest gene superfamily in mammalian genomes ( Glusman et al. 2001 ; Zozulya et al. 2001 ; Young and Trask 2002 ; Zhang and Firestein 2002 ; Olender et al. 2003 ). OR genes are organized in clusters ( Trask et al. 1998 ; Young and Trask 2002 ) and in humans are found on every chromosome save the Y and 20 ( Glusman et al. 2001 ; Zozulya et al. 2001 ). On the basis of sequence similarity, they are classified into two major classes and 17 families ( Glusman et al. 2001 ). All OR genes have an approximately 1 kb coding region that is uninterrupted by introns ( Ben-Arie et al. 1994 ; Gilad et al. 2000 ). Interestingly, approximately 60% of human OR genes carry one or more coding region disruptions and are therefore considered pseudogenes ( Rouquier et al. 1998 ; Glusman et al. 2001 ; Zozulya et al. 2001 ). In nonhuman apes, the fraction of OR pseudogenes is only approximately 30% ( Gilad et al. 2003 ). However, both humans and other apes have a significantly higher fraction of OR pseudogenes than do the mouse or the dog (approximately 20%) ( Young et al. 2002 ; Zhang and Firestein 2002 ; Olender et al. 2003 ). Thus, there has been a decrease in the size of the intact OR repertoire in apes relative to other mammals, with a further deterioration in humans ( Rouquier et al. 2000 ; Gilad et al. 2003 ). Although the causes are unclear, it seems reasonable to speculate that the high fraction of OR pseudogenes in apes reflects a decreased reliance on the sense of smell in species for whom auditory and visual cues may be more important (e.g., Dominy and Lucas 2001 ). We were therefore interested in investigating whether the high fraction of OR pseudogenes is characteristic of primates as a whole and, if not, to pinpoint when the proportion of OR pseudogenes increased. To this end, we randomly selected subsets of 100 OR genes in 19 primate species, including a human, four apes, six Old World monkeys (OWMs), seven New World monkeys (NWMs) and one prosimian. We find that a decrease in the size of the intact olfactory repertoire occurred independently in two evolutionary lineages: in the ancestor of OWMs and apes, and in the New World howler monkey. Results and Discussion Owing to the high levels of DNA sequence divergence among the primate species in our sample, orthologous OR genes could not be amplified by primers designed based on human sequences ( Gilad et al. 2003 ). Instead, we used two sets of degenerate primer pairs, constructed to amplify OR genes from all of the species studied (see Materials and Methods ). We then cloned the PCR products and determined the sequences of clones until we had identified 100 distinct OR genes from each species. A danger of this approach is that degenerate primers may bind preferentially to certain OR genes, thereby resulting in a biased representation of the OR repertoire. To safeguard against this, we tested the degenerate primers on human and mouse, for which the entire OR gene repertoire is known, by using them to amplify 100 OR genes from the two species. The sample thus obtained faithfully represented the composition of the full OR gene repertoire in human and mouse with respect to the 17 OR gene families ( Figure 1 ). Moreover, the sample estimates of the fractions of pseudogenes were accurate (see Materials and Methods ; Figure 2 ). This pilot study demonstrates that the degenerate primers yield an unbiased representation of the OR gene repertoire, as measured by the family composition and pseudogene content of the human and mouse samples. Since the primers performed well both in human and a distantly related species, the mouse, there was no reason to assume that they would not do so in nonhuman primate species. Figure 1 Results of the Pilot Study in Human and Mouse The percentage of OR genes from each family is given for the entire repertoire (filled bars) and a sample of 100 genes amplified using PC1 and PC2 degenerate primers (open bars). (A) OR genes in human. (B) OR genes in mouse. None of the differences between the full repertoires and the samples are significant at the 5% level. Only full-length OR genes (larger than 850 bp) were considered. Figure 2 The Proportion of OR Pseudogenes in 20 Species Primate species are color-coded according to family. The arrow points to the howler monkey. Datapoints (from left to right) are for apes (green): human ( Homo sapiens ), chimpanzee ( Pan troglodytes ), gorilla ( Gorilla gorilla ), orangutan ( Pongo pygmaeus ), gibbon ( Hylobates syndactylus ); for OWMs (blue): Guinea baboon ( Papio papio ), rhesus macaque ( Macaca mulatta ), silver langur ( Trachypithecus auratus ), mona ( Cercopithecus mona ), agile mangabey ( Cercocebus agilis ), black-and-white colobus ( Colobus guereza ); for NWMs (red): brown capuchin monkey ( Cebus apella ), southern owl monkey ( Aotus azarai ), spider monkey ( Ateles fusciceps ), black howler monkey ( Alouatta caraya ), squirrel monkey ( Saimiri sciureus ), wooly monkey ( Lagothrix lagotricha ), common marmoset ( Callithrix jacchus ); for one prosimian primate (brown): crowed lemur ( Eulemur mongoz ); and for the mouse ( Mus musculus ) (grey). We therefore proceeded to sequence 100 genes from 18 nonhuman primates using these primer pairs. Since the genome sequence is not available for these species, we were not able to compare the familial composition of our samples of OR genes to that of the full OR repertoires. However, with the exception of OR families 3, 11, 12, and 55 (whose absence in a sample of 100 genes is not unlikely, as they represent less than 1.8% of human OR genes), we identified OR genes from all families in all species ( Table 1 ). Moreover, the representation of the three largest OR gene families in the sample varied across species, again suggesting that there is no strong bias towards the amplification of specific families. Table 1 Distribution of OR Genes in Families across Species We then tabulated the proportion of OR pseudogenes in each species ( Figure 2 ). Consistent with previous results based on direct sequencing of full-length OR orthologs ( Gilad et al. 2003 ), we found that the proportion of OR pseudogene in the great apes and rhesus macaque is approximately 30% ( Figure 2 ). Together, these findings confirm the validity of this degenerate primer approach. We further found that the proportion of OR pseudogenes in OWMs (29.3% ± 2.4%) is very similar to that of nonhuman apes (33.0% ± 0.8%), but notably higher than that of NWMs (18.4% ± 5.6%). One NWM species, the howler monkey, was a conspicuous exception, with an elevated proportion of OR pseudogenes, similar to that of OWMs and apes (31.0%) ( Figure 2 ) and significantly higher than any other NWM (one-tailed p < 0.02 for the difference between the howler monkey and the NWM with the second highest proportion of pseudogenes, the Wooly monkey, as assessed by a Fisher's exact test [FET]). Thus, it appears that a deterioration of the olfactory repertoire occurred in all apes and OWMs as well as, independently, in the howler monkey lineage. Strikingly, a second phenotype is shared only by the howler monkey, OWMs, and apes: full (or “routine”) trichromatic color vision. In primates, trichromatic color vision is accomplished by three opsin genes whose products are pigments sensitive to short, medium, or long wavelength ranges of visible light ( Nathans et al. 1986 ). In OWMs and apes, the short-wavelength opsin gene is found on an autosome, while two distinct X-linked loci for medium and long wavelengths underlie full trichromatic color vision (and so are present in both males and females). In contrast, most NWM species carry an autosomal gene and only one X-linked gene, where different alleles encode for photopigment opsins that respond to medium or long wavelengths. Heterozygous females can therefore possess trichromatic vision, but males are dichromatic ( Jacobs 1996 ; Boissinot et al. 1998 ; Hunt et al. 1998 ). The sole exception among NWMs is the howler monkey ( Jacobs et al. 1996 ; Jacobs and Deegan 2001 ; Surridge et al. 2003 ), which has a duplication of the opsin genes on the X chromosome ( Goodman et al. 1998 ; Jacobs and Deegan 2001 ) ( Figure 3 ). Thus, full trichromatic vision arose twice in primates, once in the common ancestor of OWMs and apes and once in the howler monkey lineage. Figure 3 Phylogenetic Tree of Primates Schematic phylogenetic tree of the primate species used in the current study. Phylogenetic relationships between species are based on Harada et al. (1995 ), Page et al. (1999 ), and Surridge et al. (2003 ). Arrows indicate on which lineages the acquisition of full trichromatic color vision occurred ( Goodman et al. 1998 ; Jacobs and Deegan 2001 ). The red color highlights lineages with a high proportion of OR pseudogenes. While OWMs, apes, and the howler monkey carry 32.5% ± 6.3% OR pseudogenes in their OR gene repertoire, species without full trichromatic vision have 16.7% ± 1.0%, significantly fewer ( p < 10 −4 , or, excluding humans from the full trichromatic group, p < 10 −3 , as assessed by a Mann–Whitney U test). This p value is only indicative since the species lineages are not all independent. However, if significance is instead assessed by a FET for all pairwise comparisons of species with full trichromatic color vision and without, the difference is again striking: 94 out of 96 comparisons are significant at the 5% level. Thus, the evolution of full trichromatic vision coincided with an increase in the fraction of OR pseudogenes, indicative of a deterioration of the sense of smell. Apes and OWMs acquired trichromatic color vision approximately 23 million years ago ( Yokoyama and Yokoyama 1989 ), while the duplication of the opsin genes in the howler monkey occurred approximately 7–16 million years ago ( Jacobs 1996 ; Cortes-Ortiz et al. 2003 ). In spite of this difference in timing, the proportion of OR pseudogenes in species from both lineages is very similar. We estimated the rate of fixation of neutral gene disruptions for OR genes to be approximately 0.12 per gene per million years (Y. Gilad, S. Pääbo, and G. Glusman, unpublished data). This estimate implies that both apes, OWMs and the howler monkey could have a much higher proportion of OR pseudogenes than observed (data not shown), indicating that the process of functional OR gene loss has decreased or stopped in these species. A plausible explanation for the similar proportion of OR pseudogenes in the different lineages is that while full trichromatic vision relaxed the need for a sensitive sense of smell, it did not render olfaction unnecessary. Accordingly, while some OR genes can accumulate coding region disruptions, others are still evolving under evolutionary constraint. This model predicts that the possession of full trichromatic color vision alone allows for the loss of some but not all OR genes. A natural next step would then be to identify which OR genes or families were lost after the acquisition of full trichromatic vision. The answer to this question awaits sequence from a large number of orthologous OR genes. In this respect, it is interesting to note that the TRP2 gene, a major component of the vomeronasal pheromone transduction pathway, was found to be intact in several NWM species, but is a pseudogene in OWMs and apes ( Liman and Innan 2003 ; Zhang and Webb 2003 ). The authors raised the possibility of a connection between the acquisition of full trichromatic color vision and decreased pheromone perception, based on the difference between OWMs and apes on the one hand and NWMs on the other ( Liman and Innan 2003 ; Zhang and Webb 2003 ). However, since many traits can potentially be mapped to the lineage that leads to OWMs and apes, the connection between full trichromatic vision and pheromone perception was tenuous. Furthermore, Liman and Innan (2003 ) did not find a coding region disruption in four exons of TRP2 in the howler monkey. An intact TRP2 gene in the howler monkey would be inconsistent with the hypothesis that the enhancement of color vision replaced pheromone signaling in primates. In contrast, in the present study, we find that the deterioration of the olfactory repertoire occurred concomitant with the evolution of full trichromatic vision in two separate primate lineages. Thus, although at this point we are unable to demonstrate that the decline in the sense of smell is a direct result of the evolution of color vision, our results strongly suggest an exchange in the importance of these two senses in primate evolution. Future studies of the sensory cues involved in detection and selection of food (e.g., Smith et al. 2003 ), or the choice of a mate, may test this association directly. Materials and Methods Design and test of degenerate primers. OR genes have a coding region that is approximately 1 kb long and contains no introns. In order to test the performance of degenerate primers, we sequenced 30 genes amplified with each primer pair in human and mouse and compared the composition of the different OR families in the sample to that of the full OR gene repertoire of these two species ( Glusman et al. 2001 ; Zhang and Firestein 2002 ). We also compared the sample estimates of the proportion of pseudogenes to the proportion in the entire OR repertoire of human and mouse. Since the degenerate primers amplify only 670 bp of the approximately 1 kb coding region of the OR gene, a subset of the coding region disruptions will fall in segments of OR genes not amplified by our primers. As a result, the true fraction of OR genes carrying coding region disruptions will be underestimated by our approach. We therefore determined the proportion of OR genes with at least one disruption within the corresponding 670 bp in the entire human and mouse OR gene repertoires (47.7% and 16.3% in humans and mouse, respectively). We first tested an existing set of primers, used by Rouquier et al. (2000 ), but found significant deviations from the family composition of the full OR repertoire in both species. As an illustration, among the 60 OR genes obtained in humans, 36.6% were of the subfamily 7E (all pseudogenes), significantly more than expected given the true proportion of the 7E subfamily in the full human OR gene repertoire (12.4%, p = 2 × 10 −6 , assessed by FET). As a consequence of these biases, estimates of the proportion of pseudogenes in human and mouse obtained with these primers ( Rouquier et al. 2000 ) differ significantly from the true value ( p < 0.01, assessed by FET). We proceeded by designing new pairs of degenerate primers for the OR gene family by using the program HYDEN ( Fuchs et al. 2002 ; Linhart and Shamir 2002 ). The first primer pair, PC1 (PC1–5′: CTSCAYSARCCCATGTWYHWYTTBCT, PC1–3′: GTYYTSAYDCHRTARAYRAYRGGGTT), was designed based on class 1 human OR sequences only. The second primer pair, PC2 (PC2–5′: YTNCAYWCHCCHATGTAYTTYTTBCT, PC2–3′: TTYCTNARGSTRTAGATNANDGGRTT), was designed based on solely class 2 human OR sequences, excluding all genes that belong to subfamily 7E. Both primer pairs were designed to amplify a 670-bp product that approximately covers the region from transmembrane domains 2–7 of the OR protein. As a first step, we used each primer pair to amplify and sequence (see below) 30 genes from human genomic DNA. We found that PC1 primer pairs amplify OR class 1 and OR class 2 genes in roughly equal proportions. PC2 primer pairs amplified only OR class 2 genes, including members of the 7E OR subfamily. Based on the OR family composition that we observed for the 60 genes, we estimated that if we constructed a sample containing 25% of genes amplified with PC1 and 75% of genes amplified with PC2, we would obtain an unbiased representation of the familial composition of the human OR gene repertoire. This approach was validated by amplifying and examining 100 genes collected in the same way from human as well as from mouse. PCR and DNA sequencing Each primer pair was used to amplify a set of eight reactions in each species using a temperature-gradient PCR. The use of several annealing temperatures for each species yielded a greater diversity of amplified OR genes. PCR was performed in a total volume of 25 μl, containing 0.2 μM of each deoxynucleotide (Promega, Madison, Wisconsin, United States), 50 pmol of each primer, 1.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris (pH 8.3), 2 U of Taq DNA polymerase, and 50 ng of genomic DNA. Conditions for the PCR amplification from all species were as follows: 35 cycles of denaturation at 94°C, annealing at a gradient temperature of 48°C to 60°C, and extension at 72°C, each step for 1 min. The first step of denaturation and the last step of extension were 3 min each. The PCR products were separated and visualized in a 1% agarose gel. From each amplification set (a given primer pair in a given species), all successful products were mixed and subjected to cloning using a TA cloning kit (Boehringer, Mannheim, Germany). Cloning was followed by a touchdown PCR using the vector primers for amplifications from isolated bacterial colonies. Products were purified using the High Pure PCR Product Purification Kit (Boehringer). Sequencing reactions were performed in both directions on PCR products, using the vector primers and the dye-terminator cycle sequencing kit (Perkin Elmer, Wellesley, Massachusetts, United States) on an ABI 3700 automated sequencer (Perkin Elmer). Sequence analysis After base calling with the ABI Analysis Software (version 3.0), the data were edited and assembled using the Sequencher program, version 4.0 (GeneCodes Corporation, Ann Arbor, Michigan, United States). Assembly of the clones was done using a similarity cutoff of 98%. This cutoff ensures that Taq-generated mutations that may have been sequenced in individual clones are not counted as independent genes. Clones that were collapsed to the same contig by the assembly process were counted as one gene. Once 25 and 75 genes (independent contigs) were identified from PC1 and PC2 primer pairs, respectively, a majority consensus was generated for each gene. In order to confirm that only OR genes were amplified from all the species, we used the consensus sequences of all genes from all species as queries in a BLAT search against the human genome sequence ( http://genome.ucsc.edu/ ). In every case, the best hit was a human OR gene. This analysis was also used to insure that none of the genes were an artifact of (“jumping”) PCR fusion. Finally, each consensus sequence was searched for an uninterrupted open reading frame (ORF) in all six possible frames. If an uninterrupted ORF was found, the gene was annotated as intact. If no ORF was identified, the gene was annotated as a pseudogene. This approach probably results in an underestimate of the proportion of pseudogenes, as not all OR genes with an intact coding region are functional. Mutations in promoter or control regions of OR genes may lead to reduced or no expression. Similarly, radical missense mutations in highly conserved positions of the OR protein may result in dysfunction ( Menashe et al. 2003 ). Although it is known that there are several highly conserved positions among OR genes, it is not always straightforward to ascertain which, if any, of these positions is necessary to retain function. We therefore chose the most straightforward definition of a pseudogene: a gene without a full ORF. Supporting Information Accession Numbers Sequences for all OR genes from all primate species were deposited to GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) as accession numbers AY448037–AY449380 and AY454789–AY455274. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC314465.xml |
439009 | Genomic Analysis of Retinal Development in the Mouse | null | The eyes may be the window to the soul for poets, but for neuroscientists, they serve a more practical purpose. Of the 100 trillion or so cells that make up the human body, over 100 billion are dedicated to the structure and operation of the brain alone. Given the molecular and functional complexity inherent in such numbers, neuroscientists have historically focused on a more tractable system, the vertebrate retina, to study central nervous system development and physiology. Cells in the retina are packaged into highly ordered anatomical layers, based on their specialized functions. This organizational structure is characteristic of other regions of the central nervous system, and allows the brain to take in and integrate sensory information simultaneously, using discrete computational units. Creating such functional microprocessors depends on making the right cell at the right place and time. During development, cells undergo periods of proliferation and increasing specialization (differentiation), generating seven types of retinal cells (six types of neurons and one glial cell type) in a precise order at specific times. Mature, specialized cells arise from a pool of proliferating progenitors—cells that have already committed to becoming a retinal cell but haven't yet settled on a particular cell type. But progenitors are not all alike; they display intrinsic differences in their “competence” to produce a particular subset of retinal cells at a particular stage of development. These differences may help ensure that ganglion cells, for example, are established before photoreceptors, since photoreceptors rely on ganglion cells to transmit their signals to the brain. Classic drawing of the retina by Ramón y Cajal Which path a cell ultimately chooses stems from a combination of both intrinsic competence factors—likely determined by a cell's gene expression program—and external signals from the cell's environment. Progenitors give rise to “postmitotic” cells (cells that have exited the cell cycle and ceased proliferating), which go on to express characteristics associated with a specific cell type. Beyond this framework, the molecular underpinnings of retinal development remain obscure. Differentiated cells exhibit a gene expression program unique to their cell type, but it's not clear what accounts for underlying differences among progenitors, for example, or what factors usher retinal cells into their respective specialties. To map the genetic landscape of retinal development, Constance Cepko and colleagues looked for genes expressed in retinal cells passing through various competence levels and making cell fate choices. They determined gene expression profiles by collecting bits of gene transcripts from the retinal tissue of developing mice at two-day intervals, starting with mice entering neurogenesis and ending with mice about six and a half days old. They also collected gene expression data from postnatal day 10 and from adult mice. The authors then examined the cellular expression patterns of 1,051 of the genes that showed dynamic patterns by genomic expression profiling. Cepko and colleagues then pegged these genes to specific cell types to create a “molecular atlas of gene expression in the developing retina.” (Though the retina has many millions of cells, different cell types can be easily identified based on their telltale shape and position in the retina.) Nearly every gene known to direct retinal cell differentiation was detected in this analysis and showed high levels of expression. Genes required for cell fate choices showed peak expression near or after cells exited the cell cycle, supporting the idea that similar controls operate to put the brakes on cell proliferation and to determine cell fate. Many uncharacterized genes were expressed only in certain progenitor subsets, making them good candidates as cell fate determinants for different subtypes of retinal cells. A promising list of candidate genes for retinal development and function appear in this molecular atlas, along with candidates for retinal disease. Since many degenerative retinal diseases stem from defects in development, these genes will help researchers focus their search for therapies. And if the eye truly is the window of the nervous system, these findings may suggest general principles of cell fate determination for the developing brain, spinal cord, and other regions of the vertebrate nervous system. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC439009.xml |
521691 | Protection of pulmonary epithelial cells from oxidative stress by hMYH adenine glycosylase | Background Oxygen toxicity is a major cause of lung injury. The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include hOgg1 , hMYH , hNTH and hMTH. Methods The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or H 2 O 2 . Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05. Results Cell killing assays demonstrated cells over-expressing hMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O 2 toxicity did lead to a reduced endogenous expression of hNTH in A549 cells. Conclusion Increased expression of the DNA glycosylase repair enzyme hMYH in A549 cells exposed to O 2 and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O 2 and its metabolites. | Background Oxidative stress leading to the overproduction of free radicals in the lungs is present in many clinical situations. Such clinical settings include acute respiratory distress syndrome (ARDS), infants of prematurity going on to develop bronchopulmonary dysplasia (BPD), pathogenesis of chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, ischemia-reperfusion injury, drug-induced lung toxicity, cancer and aging [ 1 - 4 ]. Although the use of oxygen may be clinically indicated in hypoxemic situations, one must consider the potential long-term toxic side effects. For example, we know that oxygen creates cellular damage by a variety of mechanisms. Normal cellular metabolism of oxygen involves the transfer of electrons from NADH to O 2 molecules to form water (H 2 O). At normal partial pressure, 95% of oxygen molecules (O 2 ) are reduced to H 2 O and 5% are partially reduced to toxic byproducts by normal metabolism in the mitochondria [ 5 ]. These metabolites include the superoxide anion (O 2 - ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals ( • OH) all of which make up what are known as Reactive Oxygen Species (ROS) [ 6 ]. Exposure to conditions of hyperoxia as well as ionizing radiation (IR) leads to increased amounts of these ROS and their damaging effects. ROS are known to attack the lipids, proteins, and nucleic acids of cells and tissues [ 5 , 7 ]. Lipids, including pulmonary surfactant, react with ROS to produce lipid peroxides, which cause increased membrane permeability, inactivation of surfactant, and inhibition of normal cellular enzyme processes. Proteins reacting with ROS result in decreased protein synthesis due to inhibition of ribosomal translation or destruction of formed proteins. This ultimately leads to inactivation of intracellular enzymes and transport proteins resulting in impaired cellular metabolism and accumulation of cellular waste products. Lastly, ROS cause damage to nucleic acids by leading to modified purine and pyrimidine bases, apurinic (AP) /apyrimidinic sites, and DNA protein cross-links which can lead to single strand breaks [ 8 ]. Several defense mechanisms exist to combat the damaging effects of ROS. Intracellular enzymatic systems include superoxide dismutase which eliminates the superoxide anion, catalase which catalyzes the reduction of H 2 O 2 directly to H 2 O without the production of the hydroxyl radical, and glutathione peroxidase which directly reduces H 2 O 2 and lipid peroxides. Free radical scavengers, which stop free radical chain reactions by accepting electrons, include α-tocopheral (vitamin E), ascorbic acid (vitamin C), niacin (vitamin B), riboflavin (vitamin B 2 ), vitamin A, and ceruloplasmin [ 1 , 2 , 9 ]. These systems usually provide enough protection against oxygen metabolism under normal conditions, but may become depleted under conditions of increased oxidative stress [ 7 , 10 ]. The defense mechanism of interest in this paper involves the repair of oxidative damage through the human DNA base excision repair pathway (BER). BER is the most important cellular protection mechanism that removes oxidative DNA damage [ 11 ]. Damaged bases are excised and replaced in a multi-step process. Lesion-specific DNA glycosylase repair genes initiate this process. After removal of the damaged base, the resulting AP site is cleaved by AP-endonuclease generating a 3'OH and 5'deoxyribose phosphate (dRP). β-polymerase, which possesses dRPase activity, cleaves the dRP residue generating a nucleotide gap and then fills in this single nucleotide gap. The final nick is sealed by DNA ligase [ 12 - 14 ] (Figure 1A ). Figure 1 Base excision repair pathways for Oxidative DNA damage. (A) BER pathway demonstrating repair of 8-oxoG by the repair enzymes hOgg1 and hNTH . (B) hOgg1 , hMYH , and hMTH and their respective repair function. The oxidative repair genes that we have analyzed in this study include 8-oxoguanine DNA glycosylase ( hOgg1 ), human Mut Y homologue ( hMYH ), human Mut T homologue ( hMTH ), and endonuclease III ( hNTH ) all of which are present in human cells and involved in the protection of DNA from oxidative damage. The repair enzyme hOgg1 is a purine oxidation glycosylase that recognizes and excise 8-oxoguanine lesions (GO) paired with cytosine. GO can pair with both cytosine and adenine during DNA replication [ 15 ]. If repair of C/GO does not occur, then G:C to T:A transversions may result [ 5 , 15 - 17 ]. The repair enzyme hMYH is an 8-oxoguanine mismatch glycosylase that removes adenines misincorporated opposite 8-oxoG lesions that arise through DNA replication errors [ 5 , 18 - 20 ]. The repair enzyme hMTH hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-dGTP, 8-oxo-GTP, 8-oxo-dATP, and 2-hydroxy-dATP, effectively removing them from the nucleotide pool and preventing their incorporation into DNA (Figure 1B ) [ 21 ]. Lastly, the repair gene endonuclease III ( hNTH ) is a pyrimidine oxidation and hydration glycosylase that recognizes a wide range of damaged pyrimidines [ 22 ]. hNTH has also been shown to have a similar DNA glycosylase/AP lyase activity that can remove 8-oxoG from 8-oxoG/G, 8-oxoG/A, and 8-oxoG/C mispairs [ 23 , 24 ]. Subsequent steps following hNTH are identical to those following hOgg1 (Figure 1A ). A previous study has shown that over-expression of the DNA repair gene hOgg1 leads to reduced hyperoxia-induced DNA damage in human alveolar epithelial cells [ 25 ]. The primary goal of our present study was to compare the protective effects of the four main lesion-specific DNA glycosylase repair genes by individually over-expressing each in lung cells and determining which of these provides the greatest degree of protection under conditions of increased oxidative stress. Methods Cell Culture The human alveolar epithelial cell line A549 (58 year old Caucasian male), was purchased from ATCC Cat No CCL-185. The cells were grown in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco, Grand Island, NY). Passaging of cells was performed every 3–4 days with cells grown to 80% confluency in a 10 cm cell culture dish (Corning Incorporated, Corning, NY). Cells were kept at 37°C in a humidified, 5% CO2 incubator. Retroviral Vector Construction The retroviral vector pSF91.1, a gift from Dr. C. Baum from the University of Hamburg in Germany, was constructed with an internal ribosome entry site (IRES) upstream to the gene expressing enhanced green fluorescent protein (EGFP) as previously described [ 26 ]. Four DNA repair genes were individually ligated into the retroviral vector pSF91.1. hOgg1-6pcDNA3.1 was initially amplified by PCR by primers to introduce a kozak sequence at the 5' end [ 27 ]. Digestion of this product with EcoRI and SalI was performed and then hOgg1 was subcloned into digested plasmid vector pSF91.1, with T4 DNA ligase. DNA sequencing was performed to confirm integrity of the hOgg1 gene. hMYH/PGEX4T-1 and hMTH/PGEX4T-1 hMYH was a gift from Dr. A. McCullough (University of Texas Medical School, Galveston, TX) and hMTH was cloned in Dr. Kelley's lab. Plasmid DNA was prepared as above by digestion with EcoRI and SalI and ligated into pSF91.1 as above and sequencing was performed to confirm integrity of the genes. PGEX-6PI-hNTH1-wild type this gene was a gift from Dr. S. Mitra (University of Texas Medical School, Galveston, TX). Digestion with BamHI and SalI was performed and the hNTH1-wt fragment was ligated into the empty plasmid vector PUC18. The hNTH1-wt fragment was then excised with both sides flanked by EcoRI restriction sites and ligated into pSF91.1. Proper orientation of the gene was confirmed and sequencing was performed to determine the integrity of the gene. Retroviral Production and Infection DH5α competent cells (Life Technologies, Gaithersburg, MD) with each of the five DNA repair genes were grown in LB-broth with ampicillin (Sigma, St. Louis, MO). Plasmid DNA was prepared and used to transfect phoenix amphotropic cells, from the Nolan Lab (Stanford University Medical Center, San Francisco, CA), grown to ~80% confluency. On the second day sodium butyrate was added to each plate and incubated at 37°C for 6 hours. Fresh DMEM supplemented with FBS and penicillin/streptomycin was added and the plates were incubated at 33°C. Viral supernatant was collected 24 and 48 hrs later, filtered through a 0.45 μm acrodisc syringe filter (Pall Corporation, Ann Arbor, MI) and frozen at -80°C for later use. Retroviral titers were determined by fluorescent-activated cell sorter (FACS) analysis. Titers of viral supernatant were 8 × 10 5 to 1.2 × 10 6 particles/ml [ 26 ]. 2.5 × 10 5 A549 cells were suspended with the viral supernatant and plated in 1 well of a 6-well plate along with polybrene (Sigma, St. Louis, MO). This exposure was performed 6 hours per day for three days. At approximately five days from the beginning of the infection, the infected cells were analyzed using flow cytometry and sorted for EGFP expression. Western Analysis Cell pellets of sorted cells were resuspended in NuPage buffer (Invitrogen, Carlsbad, CA) and protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). 20 ug of protein were loaded into individual lanes of a NuPage Bis-Tris Gel (Invitrogen, Carlsbad, CA). The gel was then transferred to nitrocellulose paper (Osmonics Inc, Gloucester, MA). The membranes were then blocked with 1% blocking solution (Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibodies to hOgg1 (Novus Biologicals, Littleton, CO), hMTH (Novus Biologicals, Littleton, CO), hMYH (Oncogene Research Products, Darmstadt, Germany) and hNTH (Proteintech Group Inc, Chicago, IL) all at a dilution of 1:1000 except hNTH which was diluted 1:2500. They were then washed 2 times with TBST and 2 times with 0.5% blocking solution, 10 minutes per wash. The membranes were incubated with anti-rabbit secondary antibodies at 1:1000 for 1 hour at room temperature. Lastly, the membranes were washed 4 times with TBST, 15 minutes per wash. The membranes were briefly soaked in BM chemiluminescence blotting substrate (Roche Diagnostics, Indianapolis, IN) and then exposed to high performance autoradiography film (Amersham Biosciences, Buckinghamshire, England). Kodak Digital Science 1D Image Analysis software was utilized to quantify the region of interest (ROI) band mass of individual bands on films where visualized differences were detected. Hyperoxic Exposure Sorted EGFP positive A549 cells infected with the above DNA repair genes were counted and seeded into 96-well plates at a density of 1000 cells/well, 6 wells per gene. Six hours after seeding, individual plates were placed into an oxygen chamber supplied by Dr. L. Haneline (Wells Center for Research, Indianapolis, IN) located in a 37°C incubator. The oxygen chamber was then infused with 95% O 2 and 5% CO 2 . Individual plates were removed after 12, 24, 48, and 72 hours of exposure. Control A549 cells were incubated in a normal 37°C humidified-5% CO 2 incubator. O 2 concentrations were monitored with a MAXO 2 analyzer (Maxtec, Salt Lake City, UT). Four days from the beginning of the exposure, cells were assessed for cell growth/survival using the sulforhodamine B assay (SRB assay). Sulforhodamine B Assay The SRB assay (Sigma, St. Louis, MO), developed by the National Cancer Institute, provides a sensitive measure of drug-induced cytotoxicity through a colorimetric endpoint that is non-destructive, indefinitely stable, and visible to the naked eye. This assay was used to assess the cell growth/survival of over-expressed cells [ 28 ]. Cold 10% TCA was used to fix the cells to the plate. After incubation for 1 hour at 4°C, the individual wells were rinsed with water. After air-drying, SRB solution was added to each well and cells were allowed to stain for 20–30 minutes. 1% acetic acid wash was used to rinse off unincorporated dye. Incorporated dye was then solubilized in 100 μl per well of 10 mM Tris. Absorbance was measured by a tunable microplate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 565 nm. Background absorbance measured at 690 nm was subtracted from the measurements at 565 nm. Irradiation and H 2 O 2 Exposure Sorted EGFP positive A549 cells were seeded into 96-well plates at a density of 1000 cells/well. Six hours after seeding, individual plates were then exposed to radiation at doses of 250, 500, 1000, and 1500 Rads or 0.2 mM, 0.4 mM, and 0.6 mM H 2 O 2 (Sigma, St. Louis, MO). All plates including control plates were then placed into a 37°C humidified-5% CO 2 incubator. Four days after exposure, cells were fixed and assessed for cell growth/survival by the SRB assay. Natural Cell growth Sorted EGFP positive A549 cells and wild type cells were seeded individually onto four 96-well plates at 1000 cells/well. All the plates were placed into a 37°C humidified-5% CO 2 incubator. Every 24 hours for 4 days, 1 plate was removed and the cells were fixed and analyzed by the SRB assay looking at cell growth under non-toxic conditions. Growth curves and exponential growth equations were determined to look at the doubling time (DT) of cells infected with each repair gene of interest compared to vector infected and uninfected wild type cells. Statistics All drug exposure experiments were performed at least three times and individual drug doses included 6–8 wells for each group of infected cells. Analysis of cell growth and exponential growth equations were determined using Microsoft Excel. All experiments involving drug exposures were normalized to the zero dose. Data are expressed as means ± SE. The significance of differences were calculated using the paired Student's t test with significance being accepted for p < 0.05. Results Retroviral Constructs The DNA repair genes hOgg1 , hMYH , hMTH , and hNTH were ligated into the retroviral vector pSF91.1 (figure 2 ). This vector, derived from a murine stem cell virus backbone, along with each individual repair gene, was used for transfection of phoenix amphotropic cells. Viral supernatant was then collected and used to stably infect A549 cells. A heterogeneous population of A549 cells expressing EGFP was sorted so all cells used for experiments contained the genes of interest integrated into their DNA (data not shown). Figure 2 Retroviral vector pSF91.1. Depiction of the retroviral vector utilized in these experiments demonstrating restriction sites and location of entry of the gene of interest between the LTR and the IRES. Repair Gene Expression Western blot analysis was performed on sorted cells in order to verify over-expression of the four genes of interest. hOgg1 , hMYH , hMTH , and hNTH were all detected at their correct position on western blots (data not shown). Western analysis was also utilized to assess whether over-expression of each individual repair gene resulted in altered endogenous expression of the other repair genes under both non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad). Cells over-expressing the repair genes hOgg1 , hMYH , hMTH , and hNTH did not lead to altered expression of the other endogenous repair genes under the above conditions when compared to each other or pSF91.1 vector control cells (Figure 3A,3B,3C and 3D ). hOgg1 's endogenous expression was below the level of detection. The pattern of endogenous expression of hNTH was consistent for each condition when comparing cells over-expressing hOgg1 , hMYH , hMTH , and pSF91.1. Reduced expression of hNTH after exposure to 95% O 2 was noted. Figure 3 Western analysis of A549 cells over-expressing individual repair genes and effect on endogenous glycosylase level. (A) Endogenous expression of hOgg1 was not altered in A549 cells over-expressing any of the other repair genes when analyzed after non-toxic and toxic exposures. hOgg1 protein was not detectable for any of the cells under the above conditions when compared to cells over expressing hOgg1 . (B) and (C) Endogenous expression of hMTH and hMYH respectively also were not altered in A549 cells over-expressing any of the other repair genes when analyzed after non-toxic and toxic exposures. (D) Endogenous expression of hNTH was analyzed under non-toxic and toxic conditions in A549 cells over-expressing the other repair genes. Reduced expression of hNTH was observed equally with all of the other genes after exposure to 95% O 2 . Endogenous expression of all four genes was equivalent under the above conditions in vector control cells; pSF91.1 (data not shown). Lastly, we assessed endogenous expression of each individual repair gene in cells infected with pSF91.1 following non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad) at 24 and 48 hrs after the onset of exposure. Endogenous hMYH and hMTH were expressed to the same degree. hOgg1 's endogenous expression was below the level of detection using western analysis (results not shown). When analyzing endogenous hNTH expression, it was noted that hyperoxia at 24 hrs and 48 hrs resulted in reduced protein expression by 93% and 64% respectively. There also was a small increase in expression of hNTH noted after 1000 Rad one day post exposure that was back to baseline by two days post exposure. ROI band mass quantification demonstrated this finding (Figure 4A and 4B ). Two or more replicates were performed for each western analysis to determine consistency of the results. Figure 4 Western analysis of endogenous hNTH repair gene after exposure to O 2 and IR. (A) Analysis of hNTH expression in A549 vector control cells following O 2 or IR treatment. The ROI band mass mean intensity was calculated for individual bands and hNTH expression was normalized to the corresponding actin loading control. (B) Graph of ROI band mass normalized to the pSF91.1 zero dose. Protection from Hyperoxia and Radiation A549 cells expressing hMYH demonstrated increased survival after exposure to conditions with elevated levels of oxygen compared to cells expressing only the pSF91.1 vector (Figure 5A ). Results were highly significant at all time points except after 12 hours O 2 where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12% after 12 hours O 2 exposure to 7% after 72 hours O 2 exposure. A549 cells expressing hMYH also demonstrated increased survival after exposure to all doses of radiation in comparison to pSF91.1 (Figure 5B ). These results were also highly significant at all doses of radiation except at 250 Rads where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12%–14% for all doses of radiation. Also noted in these experiments was that vector control cells demonstrated no significant difference in survival at all doses of O 2 and radiation in comparison to wild type A549 cells. Figure 5 Cell survival analysis following O 2 , IR, and H 2 O 2 treatments. A549 cells over-expressing hOgg1 , hMYH , hMTH and hNTH following (A) O 2 , (B) IR, and (C) H 2 O 2 . Brackets indicate statistical significance at * p < 0.05 and ** p < 0.001 compared to pSF91.1 at each individual dose for 1 representative experiment. Experiments looking at the effects of H 2 O 2 on cells expressing the repair genes did not demonstrate increased survival for any of these repair genes when compared to vector control cells (Figure 5C ). This data demonstrates that over-expression of hMYH has the ability to improve cellular survival under conditions of hyperoxia and radiation but may not be able to overcome the toxicity of H 2 O 2 . Cell Growth Cell growth under normal conditions was ascertained to determine if over-expression of any of the repair genes caused an alteration in the growth of cells in the absence of oxidative stress. Wild type A549 cells and cells expressing pSF91.1, hNTH , hOgg1 , and hMTH appeared to grow at similar rates with doubling times within the same range. A549 cells expressing hMYH did show a slower growth rate that resulted in significant differences in cell number by day 3. The calculated doubling time for the cells over expressing hMYH is > 3 hrs longer than the cells with the other repair genes and vector alone (Figure 6 ). This slowing of growth may allow for more time to repair DNA damage, ultimately leading to increased cell survival following oxidative stress. Figure 6 Cell growth curve and associated doubling times (DT). A549 cells over-expressing hMYH grow at a slower rate in comparison to all other cells under non-toxic conditions resulting in a prolongation of the doubling time. Of note, all other over-expressed cells have approximately the same doubling time as wild type A549 cells. Statistical significance noted at ** p < 0.001 compared to pSF91.1 for 1 representative experiment. Discussion Oxidative stress to the lung leads to cellular DNA damage as evidenced by the release of specific gene products known to regulate DNA base excision repair pathways such as p53 and p21 [ 29 - 31 ]. Alterations in pro-inflammatory mediators, transcription factors, and other related gene products are also observed [ 32 ]. This injury has been shown to be associated with features of both cellular necrosis and apoptosis [ 33 - 35 ]. The resultant cellular inflammation and death from oxidative stress has a dramatic impact on the outcome of patients in the clinical setting [ 7 , 36 ]. Most of our current clinical therapy towards oxidative stress in the lung involves both supportive measures and prevention. Research dealing with oxidative lung injury has focused mainly on enhancing antioxidant enzymatic processes and free radical scavengers [ 37 - 40 ]. The ability to alter cellular survival by increasing specific DNA repair mechanisms may add another approach to the treatment of oxidant-mediated lung injury. Many investigators have used hydrogen peroxide as a substitute for hyperoxia since it is known to be one of the metabolites produced by the metabolism of oxygen. ROS such as H 2 O 2 and those produced by hyperoxia clearly lead to DNA damage but questions exist as to whether H 2 O 2 leads to the same deleterious effects upon DNA as hyperoxia. Analysis of our growth curves after exposure to H 2 O 2 in comparison to hyperoxia and IR clearly indicates that cellular protection by oxidative DNA repair genes is specific to the agent used. Because no protection was observed with over-expression of any of the repair genes following exposure to H 2 O 2 , we speculate that the damage it causes is dissimilar. It may be that its damage not only involves oxidized bases, but may also include other forms of DNA, lipid, and protein damage that are not corrected by oxidative DNA repair genes. Alternatively, the amount and type of damage evoked by H 2 O 2 could be beyond that which can be corrected by over-expressing these repair genes. Another form of stress known to induce damage through the formation of ROS is IR. Radiation induced free radical damage to DNA has substantial overlap with that of oxidative damage [ 41 - 43 ]. The protection provided by specific oxidative DNA repair genes under conditions of IR, was notable throughout our experiments only with the repair enzyme hMYH . The primary agent utilized to induce the formation of ROS was an oxygen rich environment. The use of oxygen as a stressor leading to the formation of ROS, offers a distinct advantage over IR and H 2 O 2 by mimicking the clinical situation where constant exposure to hyperoxia leads to cumulative cellular damage which further compromises repair. We determined that survival of A549 cells was also enhanced to a small degree with increased expression of the repair enzyme hMYH . This was an unexpected finding as we anticipated the repair gene hOgg1 would demonstrate the greatest protection in response to oxidative stress based on previous studies, however these experiments utilized the colony forming assay (CFA) to detect improvements in survival [ 25 ]. Additionally, the CFA may provide different results compared to the SRB assay, which allows for growth analysis over a shorter window of time. Furthermore, their study did not look at the repair enzyme hMYH and its impact on survival. Another study has investigated the repair function of hMYH in MYH-deficient murine cells. It was demonstrated that transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:GO repair [ 44 ]. An interesting observation noted while doing our experiments lead us to look at individual growth characteristics of cells over-expressing each of the oxidative repair enzymes. Cells over-expressing the repair enzyme hMYH clearly grow at a slower rate when compared with the other enzymes. The mechanism behind this is not understood at this point in time. The repair action of hMYH is known to remove adenines misincorporated opposite 8-oxoG lesions. This lesion occurs when a C/GO lesion is allowed to replicate before being corrected by hOgg1 . Repair by hMYH is not a final corrective measure. The product of hMYH activity is the lesion C/GO, which allows hOgg1 to have another opportunity to remove 8-oxoG opposite cytosine. We know that A549 cells possess the hOgg1 gene based on a previous study demonstrating the presence of this gene after amplification by genomic PCR [ 45 ]. We also have demonstrated endogenous activity of hOgg1 in A549 cells by using an 8-oxoguanine bioactivity assay. Therefore, our explanation of these results is that the slowed growth created by hMYH may provide a wider window of opportunity for the repair process to take place, which ultimately grants endogenous hOgg1 another opportunity to remove the 8-oxoG lesion created by oxidative stress. As noted in the methods section, the SRB assay provides a sensitive measure of drug-induced cytotoxicity that is used to assess cell proliferation/survival. The reduced cell proliferation of A549 cells over-expressing hMYH under non-toxic conditions may likely underestimate the magnitude of the protective effect of this particular repair enzyme. This may in fact make the results even more significant. Recent studies have discovered hereditary variations of the glycosylase hMYH that may predispose to familial colorectal cancer [ 46 , 47 ]. Others have looked for hMYH variants in lung cancer patients and have not identified any clear pathogenic biallelic hMYH mutations or an over-representation of hMYH polymorphisms [ 47 ]. The A549 cell line has not demonstrated somatic mutations in hMYH , but a single nucleotide polymorphism (SNPs) has been noted [ 45 ]. The impact on function by this SNP is unknown. It would appear that the function of hMYH is very important in preventing somatic mutations leading to cancer in the gastrointestinal tract. Although studies to date have not demonstrated this same relationship with lung cancer, we do know that the lungs are subjected to large quantities of ROS under certain conditions as discussed earlier. The formation of mutations from oxidative stress does have other deleterious effects on cells including cellular death by necrosis and apoptosis. Tissue viability is dependent upon mutation correction and replication of the surviving cells to replace those that have died. The ability to enhance cellular survival, after specific oxidative exposures, is evident after increased production of the hMYH repair gene in these experiments. We additionally wanted to determine the level of endogenous expression of the glycosylase repair genes in the pulmonary epithelial A549 cell line. Others have demonstrated how different stressors lead to alterations in the endogenous production of specific repair genes. For example, it has been shown that endogenous gene expression of hOgg1 was elevated following exposure to crocidolite asbestos which is known to cause an increase in 8-oxoG levels [ 48 ]. It has also previously been reported that treatment of A549 cells with sodium dichromate, a pro-oxidant, leads to a reduction of hOgg1 protein expression that was not observed with H 2 O 2 [ 49 ]. One additional study demonstrated a dose dependent down regulation of hOgg1 protein expression in rat lung after exposure to cadmium, a known carcinogen associated with the formation of intracellular ROS [ 50 ]. In our experiments we were able to demonstrate that both hyperoxia and IR do not appear to impact the endogenous expression of hOgg1 , hMYH , and hMTH at 24 and 48 hours following exposure. It was noted that endogenous hNTH was reduced after hyperoxia at 24 and 48 hours after the onset of exposure. One would speculate that this reduction in endogenous hNTH secondary to hyperoxia is related to either decreased production or increased destruction in response to O 2 exposure. Over-expression of this repair enzyme did not result in improvements in survival after O 2 exposure based on our experiments. It may be that endogenous levels are adequate to correct this specific mutational burden for these experiments. Furthermore, no previous studies have determined how cells over-expressing specific repair genes may impact endogenous expression of the other oxidative BER genes under both normal and oxidative stress conditions. We were also able to demonstrate that endogenous expression of glycosylase repair genes were not altered under these conditions secondary to the over-expression of any of these genes. This is an important finding for interpretation of survival data; protection of cells is due to the over-expression of the specific gene and not due to enhancement of other endogenous repair enzyme levels, at least for the genes studied under these conditions. Some limitations may exist in using a lung carcinoma cell-line, which likely differs both in proliferative properties as well as in response to oxidative stress in comparison to primary epithelial cells. The enhanced cell growth observed with cell lines may be more reflective of undifferentiated alveolar type II cells which are likely to replace terminally differentiated alveolar type I cells after injury/death due to oxidative stress. This may not be a true reflection of growth under non-toxic conditions when very little cell division is occurring. This is an inherent problem observed when comparing cell lines with primary cells and results need to be interpreted in a way that considers this. It is difficult to know how this will translate to pulmonary epithelial cells in vivo at this stage. It certainly would appear that the protection observed is modest in degree in this pulmonary epithelial cell line. Further experiments assessing the function of the repair enzyme hMYH in this model will be important to perform in order to delineate the findings of slowed growth under normal conditions and improved survivability under conditions of O 2 and IR. More research looking at the potential for combination therapy, including DNA repair mechanisms in conjunction with other antioxidant defense mechanisms may be another approach to enhancing cell survival, which may lead to better clinical outcomes. Alternatively, cell survival may not be the most important end point for hyperoxia studies. Given that 8-oxoG, if left unrepaired, leads to G:C to T:A transversions, there may be an increase in mutational burden by these cells that isn't reflected in cell survival. Further experiments studying the impact on mutation production is underway. Ultimately, experiments need to be done in animal models to determine the translation to in vivo pulmonary cells. Conclusions In summary, we have demonstrated that over-expression of the DNA glycosylase repair enzyme hMYH may enhance survival of a pulmonary epithelial cell line after exposure to conditions of IR and hyperoxia. We have also demonstrated that over-expression of hMYH leads to a slowing of growth of A549 cells under non-toxic conditions, which may in part play a role in this enhancement of survival by providing a wider window of opportunity for repair of oxidized lesions to occur. Lastly, we demonstrated that over-expression does not lead to altered endogenous expression of these repair genes. As the understanding of DNA repair mechanisms continues to grow and the evolution of gene therapy takes place, more treatment options may be available in the clinical setting to help with many disease processes including the damaging effects of oxygen and its metabolites. List of abbreviations apurinic, AP; base excision repair, BER; Dulbecco's modified Eagle's medium, DMEM; deoxyribose phosphate, dRP; enhanced green fluorescent protein, EGFP; fetal bovine serum, FBS; hydrogen peroxide, H 2 O 2 ; ionizing radiation, IR; internal ribosomal entry site, IRES; long terminal repeat, LTR; oxygen, O 2 ; Sulforhodamine B, SRB; reactive oxygen species, ROS; region of interest, ROI; Tris-Borate-EDTA, TBE; tris-buffered saline Tween-20, TBST; 8-oxoguanine, GO and 8-oxoG Authors' contributions TK conducted the majority of the research experiments, performed the statistical analysis, and drafted the manuscript. MR conducted some of the cell survival experiments and participated in the design of the study. YX and XC helped with production of the lesion specific DNA repair genes. MK conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521691.xml |
544960 | Australia and New Zealand Health Policy: a new journal | Australia and New Zealand Health Policy is a new journal which aims to promote debate and understanding about contemporary health policy developments in Australia and New Zealand. Although there are other international journals focussing on health policy, there are no Australian or New Zealand journals with this focus. One of the aims of Australia and New Zealand Health Policy is to focus on contemporary critiques and contemporary developments. Accordingly an e-journal format is particularly appropriate. Australian and New Zealand Health Policy is an open access journal which means that all articles will be freely and universally accessible online which, amongst other things, means that all articles will be freely and universally accessible online without any barriers to access, which increases their visibility. | Editorial Welcome to Australia and New Zealand Health Policy a new journal which aims to promote debate and understanding about contemporary health policy developments in Australia and New Zealand. Health policy is regularly in the media and is a high profile issue at election times. In Australia the health system has been characterised by conflicts over values and policy choices over the decades. So pervasive is this conflict that Sax entitled his 1984 book about health services, "A Strife of Interests" [ 1 ]. Health policy in New Zealand has also had a turbulent time over the past decade [ 2 , 3 ]. Health policy changes in Australia and New Zealand are thus ripe for analysis. Australia and New Zealand Health Policy aims to provide a prestigious venue for analysis and critique of health policy in the two countries. Why a new journal? Although there are other international journals focussing on health policy, there are no Australian or New Zealand journals with this focus. Other related-area local journals are medical, public health or hospital-related. Although the local journals publish occasional policy articles, this area is not their principal interest, nor are they necessarily the journals which policy-oriented academics or policy practitioners scan to keep abreast of developments. The absence of a health policy journal serving Australia and New Zealand has long-term consequences for the development of systematic analysis of and research into health policy. One consequence is that there is no forum where health policy developments are documented and tracked, a lacuna which precludes cumulative analyses of trends. Australia and New Zealand Health Policy will address this by publishing annual reviews of policy developments. As one of the aims of Australia and New Zealand Health Policy is to focus on contemporary critiques and contemporary developments, an e-journal format is particularly appropriate. Debate will also be stimulated by providing for 'Comment' on published articles, in a way analogous to a letters column. Australia and New Zealand Health Policy is a peer reviewed journal. In keeping with its policy-applied focus, articles will be refereed by two referees, preferably one with a strong practitioner background, such as currently or recently employed in a policy role in a health authority, and one from an academic background. At least one of the referees will have substantive content knowledge relating to the article. Articles published in Australia and New Zealand Health Policy will be listed in PubMed and permanently archived in PubMed Central as well as certain other national archives. Journal scope Australia and New Zealand Health Policy contributes to understanding of health policy development and practice with a particular focus on Australia and New Zealand. It welcomes submissions which: • Review and critique contemporary health policy issues; • Identify major trends in health policy and emerging policy issues, including new evidence about the effect of policy changes; • Identify impacts of health services research on new policies; • Identify major public policy and governance trends and their application to health policy; • Analyse contemporary health policy themes which cut across a range of policy areas; • Report on international policy developments and new international comparisons of health policy involving Australia and/or New Zealand; or • Critique contemporary health policy developments. Open Access Australia and New Zealand Health Policy is an Open Access journal, which means: • All articles will be freely and universally accessible online without any barriers to access, which increases their visibility. • You and your peers will be free to print out copies of your article, email it to colleagues, and post it on the web because of the BioMed Central copyright and license agreement . Open access journals are funded by article processing charges rather than journal subscriptions. The costs are therefore borne by the authors, their institutions of from their grants. That is, all access to journals is free to readers via the web (BMC online-only journals). Authors from institutional supporters are exempt from authorship charges. The institutional supporters pay a sliding scale based on the number of staff and postgraduate students in biomedical sciences. Institutional subscription has a number of benefits. In addition to the direct benefits in terms of waived author fees, there are public policy benefits in supporting an open access journal regime such as Biomed Central. Open access journals are one mechanism for putting pressure on regular journal publishers to moderate their price increases. Unfortunately there are no New Zealand institutional subscribers to BioMed Central at present, which means that New Zealand authors will face article processing charges, although no article processing charges will be payable on manuscripts submitted in the first six months following the launch of the journal. Article processing charges are also usually regarded as a legitimate charge against research grants. In the medium term, alternative arrangements, such as institutional support, should be encouraged, although after this time the editor-in-chief will be able to grant a limited number of discretionary processing charge waivers. The first articles Australia and New Zealand Health Policy commences its publication program with a series of articles which describe and evaluate health policy developments in Australia in 2003. Responses or commentaries on these articles would be welcome. It is the aim of the Editorial Board to encourage a cluster of articles at the start of each year about health policy developments in Australia and New Zealand in the previous calendar year. Authors who have an interest in reviewing contemporary developments are encouraged to submit manuscripts on these themes early in the New Year so that the articles can contribute in a timely way to health policy debate in the two countries. S.J.Duckett Editor-in-Chief | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544960.xml |
514616 | In vitro fertilization and artificial activation of eggs of the direct-developing anuran Eleutherodactylus coqui | Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and in vitro fertilization of eggs of the Puerto Rican coqui frog obtained by hormonally induced ovulation. We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus. | Background The study of reproduction and its artificial manipulation is important in many fields. For example, in sea urchins, an animal's testes can be dissected and sperm is activated by exposure to seawater. Eggs can be released by injecting KCl into the perivisceral cavity, and mixing eggs and sperm in vitro produces fertilization, as evidenced by the appearance of the fertilization membrane and subsequent development of embryos [ 1 ]. These simple techniques have been the basis for such dissimilar studies as those of Berdishev [ 1 ], dealing with the role of fatty acids and cannabinoids in fertilization, to investigation of the gene expression patterns of hybrids by Nielsen and coworkers [ 2 ]. Artificial reproduction has also been well-studied in mammals, and cloning of eutherians from somatic cells is now common [ 3 - 8 ]. Harvested eggs can be enucleated and merged with a somatic cell and the reconstructed embryos cultured in vitro before being implanted into surrogate mothers [ 8 ]. These methods have opened up new possibilities in both basic and applied science [e.g. [ 9 ]]. Importantly, artificial fertilization has been utilized as a means of assisting with the conservation effort of declining species [ 10 , 11 ]. Frogs have been favorite model organisms in reproductive and developmental biology for many years, mainly because of the ease with which they can be kept in captivity; their external fertilization; easily visible development in large, transparent eggs; and large numbers and ease of manipulation of their eggs. Consequently, research on frogs has often been in the vanguard of advancement in artificial reproduction techniques, and much is known about a few model species such as the African clawed frog, Xenopus laevis and the North American leopard frog, Rana pipiens [e.g. [ 12 - 15 ]]. Indeed, the first vertebrate cloned from a somatic nucleus was a frog [ 16 ]. Briggs and King injected female R. pipiens with male pituitary glands to induce ovulation and deposition of unfertilized eggs. The eggs were mechanically activated by pricking with a needle, a process which brings the pronucleus immediately under the surface of the animal pole. Taking advantage of this situation, the pronuclei were extruded, along with a small amount of cytoplasm, using a glass needle. In other species, such as the Xenopus or the axolotl, UV radiation can be used to destroy the female pronucleus instead [ 17 , 18 ]. Development was then directed by a somatic nucleus microinjected into the cytoplasm of the enucleated egg. In another group of experiments, Kroll and Amaya [ 19 ] developed an effective and reliable method for creating transgenic Xenopus : testes were macerated in solution and the sperm membranes partially dissolved, allowing access to the condensed chromosomes. Linearized bacterial plasmids containing genes of interest were mixed in with the sperm solution and recombinant ligase was used to covalently insert the bacterial plasmids into the sperm genomic DNA, resulting in the insertion of many copies of the plasmid construct into each genome. These nuclei were then microinjected into mature eggs, generating, under appropriate conditions, hundreds of nonmosaic, transgenic embryos. Such techniques allow the investigation of gene function in these species [e.g. [ 20 ]]. Clearly, there are enormous advantages to being able to manipulate a species' reproduction in the laboratory. However, despite the multiplicity of studies concentrating on anurans, to date all model species are aquatic breeders. Yet amphibians have the largest diversity of breeding strategies among terrestrial vertebrates, and it is to be expected that species with different reproductive strategies will require different methods for their manipulation in the laboratory. Therefore, many species remain experimentally intractable. Notably, terrestrial-breeding frogs, a very large and diverse group of organisms, are largely inaccessible to reproductive investigations. The neotropical frog genus Eleutherodactylus is characterized by terrestrial breeding and direct development without an aquatic larval stage. With more than seven hundred described species, this is the largest vertebrate genus [ 21 , 22 ]. There has been considerable experimental attention focused on Eleutherodactylus frogs, ranging from basic developmental biology [ 23 - 26 ]; to ecology [e.g. [ 27 - 30 ]]; to the evolution of development [ 23 , 31 ]. However, there are as yet no available techniques for performing in vitro fertilization in these frogs. The development of such techniques would allow additional investigations into the genetic regulation of direct development in these species and would also assist with conservation of declining populations, an important goal considering the fact that many species of Eleutherodactylus are declining, and several are already extinct [ 32 , 33 ]. Eleutherodactylus coqui are small tree frogs with internal fertilization and direct development [ 34 ]. This species is extremely common in the forests of Puerto Rico, and it has been found that their population size is limited by the availability of retreat sites, as opposed to food resources [ 35 ]. As with all other studied Eleutherodactylus species, E. coqui embryos develop directly into tiny froglets in terrestrial eggs, without a tadpole stage [ 36 ]. Protocols for the husbandry of these frogs have been reported, and it is possible to maintain them in the laboratory for multiple generations [ 37 , 38 ]. A method has also been developed to induce ovulation using an artificial form of luteinizing hormone-releasing hormone (LHRH) [ 39 ]. It is know in this species that sperm entry occurs at a small disc at the animal pole of the egg, and that polyspermy is apparently common but does not interfere with development [ 40 ]. Cortical granules and their exocitosis have also been described using electron microscopy [ 40 ], but the large (5 mm diameter), opaque and featureless eggs make it difficult to observe the rising of a fertilization membrane. As E. coqui is arguably the best-studied terrestrial-breeding frog, we have focused on this particular species as a model for the development of reproductive techniques. Materials and Methods Adult Eleutherodactylus coqui frogs were collected in Puerto Rico near El Verde Field Station in the Luquillo mountains and transported to Tulane University where they were housed and fed as previously described [ 37 ]. All animals were handled and experiments performed in accordance with the standards outlined in the NIH Guide for the Care and Use of Laboratory Animals. Natural matings were performed by placing a gravid female and a calling male together as described [ 37 ]. Mature, unfertilized eggs were obtained by injection of gravid females with 20 μg of des-Gly, D-Ala LHRH ethylamide (Sigma, St. Louis, MO. Catalog Number: L4513), as reported [ 39 ]. Hormonally induced females were placed in plastic bags and allowed to deposit unfertilized eggs. Eggs were experimentally manipulated without moving them from the surface of the plastic bag where they were deposited. Sperm was obtained from adult male frogs that were anesthetized by immersion in 5% benzocaine solution, decapitated and double-pithed. Testes were removed by dissection and macerated with fine forceps. In vitro fertilization (IVF) experiments using sperm in solution were carried out by macerating the testes from a single frog in 500 μL of sperm dilution buffer (SDB: 10 mM NaCl, 0.2 mM KCl, 0.1 mM CaCl 2 , 0.1 mM MgCl 2 , 0.5 mM Hepes pH 7.5), and adding this dropwise over the tops of the eggs or injecting it under the jelly coat using a tuberculin syringe (28.5 gauge, 13 mm length). Alternatively, small pieces of macerated testes were placed directly on top of each egg without the use of buffer solution. Incubation of sperm with egg jelly was accomplished by vigorously vortexing the jelly from one egg in 100 μl of SDB. Sperm was then incubated for 10 min in the jelly/buffer supernatant. Sperm preparations were checked for morphology, movement and membrane integrity by fluorescent microscopy with Live/Dead sperm stain (propidium iodide and SYBR 14) (Molecular Probes, Eugene, OR) using an Olympus BH-2 microscope [ 41 ]. Additionally, sperm were stained with 1 μM Lysosensor green fluorescent dye (Molecular Probes, Eugene, OR) and imaged with a Zeiss LSM 510 META laser-scanning confocal microscope [ 42 ]. For artificial activation experiments, mature eggs were either mechanically stimulated by gentle poking with fine forceps, taking care to penetrate the jelly coat but not the plasma membrane, or immersed in a solution containing 10 mM CaCl 2 with 0.1 mM A23187 calcium ionophore. Scoring of activation was done by noting the appearance of the first cleavage furrow (see figure 1 ). Artificial activation experiments were also performed on oocytes dissected directly from the ovisac of gravid females. These oocytes were similarly treated by poking or exposure to 10 mM CaCl 2 with 0.1 mM A23187 calcium ionophore, with or without pretreatment for 12 hrs. with 3 μM progesterone [ 43 ]. Figure 1 Early egg development. A) Untouched, unfertilized egg. The surface of the egg under the jelly coat is featureless. B) Sperm-activated egg at six hours post-fertilization at the four-cell stage. Note the straight, ordered cleavage pattern (arrow). C) Artificially activated egg pseudocleaving. Note the jagged and disorganized cleavage pattern (arrow). D) An egg pseudocleaving at 16 hours. This egg was not handled and did not cleave within the first ten hours after deposition. At ten hours it was poked, and started pseudocleavage shortly thereafter (arrow). Scale bar = 1 mm. Eggs were allowed to develop at room temperature in parafilm-sealed 60 or 100 mm polystyrene petri dishes and were moistened with an antibiotic solution consisting of 25 μg/ml amphotericin B, 10 U/ml penicillin and 10 ug/ml streptomycin. Eggs were scored as successfully fertilized following neurulation (stage 2 sensu Townsend and Stewart [ 36 ]; stage 14 sensu Gosner [ 44 ]). Results and Discussion Artificial activation Table 1 shows the activation effects of either mechanical stimulation or A23187 calcium ionophore exposure on the unfertilized eggs of E. coqui . This ionophore non-specifically activates the unfertilized eggs of a variety of species through stimulation of a calcium-dependent signalling cascade [ 45 ]. Ten hours after laying, 11 of 32 eggs (34%) pseudocleaved, even if left undisturbed. As unfertilized eggs do not posses a centrosome, if they are artificially activated, the mitotic apparatus cannot form properly and divisions are irregular. This well-described process is called pseudocleavage [[ 46 ]; figure 1 ]). Activation almost doubled to 28 of 44 (64%) when the eggs were poked with forceps. Further, 30 of 36 eggs (83%) exposed to 0.1 mM calcium ionophore pseudocleaved. These percentages include both eggs that would have auto-activated –presumably 34%- as well as eggs that were activated by the mechanical or chemical treatments. Table 1 Artificial activation of E. coqui eggs Egg origin Treatment Number cleaving at 10 hours/Number tested (%) Number cleaving at 16 hours, after being poked at 10 hours/Number tested (%) Eggs laid in response to hormone treatment Undisturbed 11/32 (34) 6/8 (75) Poked 28/44 (64) - A23187 30/36 (83) - Oocytes dissected directly from ovisac Undisturbed 0/40 - A23187 0/40 - Progesterone 0/40 - Progesterone & AA23187 0/40 - To test whether the pseudocleavage response was an effect of our stimuli and not a reaction tied to other uncontrolled variables, we examined eight eggs that had remained undisturbed and that had not started pseudocleavage ten hours after laying. At this time, we poked them with fine forceps, and 6 of 8 (75%) began pseudocleaving six hours later (table 1 ). This delay in activation as a response to a delay in the stimulus is a strong indication that our manipulation is in fact responsible for eliciting the onset of cell division. An interesting observation was that one third of all eggs deposited in response to hormone treatment activated of their own accord. This may be due to the mechanical stress to which the eggs are exposed during oviposition. Clearly, mechanical stimuli are able to activate the eggs, and stress incurred in during transit from the ovisac may be sufficient to cause activation. This would presumably not affect E. coqui during natural matings because this species undergoes internal fertilization, and the eggs will have already been fertilized prior to deposition [ 34 ]. In order to examine this hypothesis, we dissected oocytes directly from a female's ovisac, circumventing the passage through the oviduct and cloaca, and attempted to activate them with calcium ionophore (table 1 ). Controls were also performed with and without progesterone pretreatment in order to induce maturation. Since there is no information on the stage at which E. coqui eggs are arrested or what signal takes them out of their arrest, we followed procedures used in Xenopus [ 43 ]. However, none of these oocytes activated, regardless of the treatment. This may be because the oocytes did not respond to treatment with progesterone, and so never matured. Another possibility is that oocytes need to receive a signal from the oviducts and/or be coated in jelly before they can mature. The ability to initiate activation after a long delay was interesting as we suspected that E. coqui eggs might be sensitive to aging, as has been reported under certain conditions for the externally fertilizing X. laevis [ 47 ]. Because E. coqui has internal fertilization, we suspected that eggs laid unfertilized might degenerate rapidly, complicating the artificial manipulation of this species' reproduction. Consequently, we investigated the ability of eggs to be activated as a function of time. When we artificially activated eggs with the calcium ionophore at different time points and examined them six hours post treatment, the percentage that pseudocleaved was high even ten hours after being laid (Figure 2 ). Twenty-four hours after deposition, however, the eggs were no longer able to activate. Thus, there is an extended period in which it is possible to carry out experiments without concern for a decrease in activation potential. Figure 2 Artificial activation of E. coqui eggs in relation to time after deposition. Unfertilized eggs were treated with calcium ionophore to induce activation at 0 (n = 12), 1 (n = 12), 2 (n = 10), 3 (n = 12), 4 (n = 12), 5 (n = 12), 10 (n = 18), and 24 (n = 18) hours. Eggs were scored for activation by the presence of cleavage furrows at 6 hours post treatment. Note that for the 10 and 24 hour time points, six eggs at each time point had already auto-activated by 6 hours post deposition. At the 10 hour time point, nine additional eggs activated later in response to ionophore treatment, while at 24 hours, no additional eggs were observed to activate after ionophore treatment. In vitro fertilization The average fertilization efficiency for natural matings conducted in our laboratory was 72% (see table 2 ). As roughly one third of all unfertilized eggs laid in response to hormone treatment auto-activate (table 1 ), and so only approximately 66% of the eggs in a given clutch will actually be receptive to sperm. If we assume these two factors to be independent -because we hypothesize that the eggs auto-activate during laying, a problem that doesn't arise in natural matings- then only 48 of every hundred eggs will be available for IVF (100*0.66*0.72). However, despite these complications, we were able to obtain an in vitro fertilization efficiency of 27% -or, rather, 56% of all receptive eggs (27/0.48) – by simply mincing the testes and adding them directly over the eggs (see table 2 ). Other IVF techniques were not as successful. Using sperm diluted in SDB resulted in only a 12% total fertilization efficiency (table 3 ). Table 2 Natural mating fertilization percentages for E. coqui in captivity. Clutch Number of eggs laid Number that developed to neurula (%) 1 39 24 (62) 2 58 36 (62) 3 30 28 (93) 4 42 34 (81) Total 169 122 (72) Table 3 In vitro fertilization of E. coqui eggs Fertilization protocol Number that developed to neurula/Number tested (%) Sperm solution dripped over the eggs 5/63 (8) Testes minced directly over eggs 8/30 (27) Sperm solution injected under the jelly coat 0/12 Sperm incubated in jelly buffer and then injected under the jelly coat 1/10 (10) Poked, then fertilized 15 minutes later by mincing testes directly over the eggs 0/36 Sperm concentration may play a role in fertilization efficiency as the use of diluted sperm resulted in decreased fertilization. In support of this possibility, polyspermy has been observed in this species and is apparently not deleterious to fertilization and development [ 40 ]. A second possibility is that fertilization efficiency is linked to sperm capacitation and acrosome reaction. We attempted to study this possibility by examining the acrosomes of fresh and treated sperm using Lysosensor green fluorescent dye (Molecular Probes, Eugene, OR). This dye concentrates in low pH vesicles of living cells through an unknown mechanism and was shown to accumulate and preferentially stain the acrosome in X. laevis sperm [ 42 ]. However, we were unable to observe acrosome-specific staining in E. coqui sperm using this dye (data not shown). We also examined the possibility that a component of the egg jelly coat may be important for sperm capacitance. To test this, we incubated sperm with SDB and jelly, or SDB alone, and injected this under the jelly coat of eggs. None of 12 eggs were fertilized by sperm incubated with SDB alone, while pre-incubation of sperm with an extract of the jelly coat in SDB resulted in one fertilization out of 10 eggs (10%). This is considerably less than the 27% efficiency following direct placement of minced testes over the eggs, but suggests that interactions between sperm and the jelly coat may play a role in sperm capacitance and subsequent fertilization. To test the functional response of the eggs, we attempted to fertilize artificially activated eggs. As was explained above, we were able to achieve an IVF success rate of 27%. However, if we poked the eggs fifteen minutes prior to direct fertilization, none (0/36) developed (see table 3 ). If we assume our expected fertilization rate to be 25%, the possibility of this result being due to chance is (1-0.25) 36 = 3.2 × 10 -5 , or less than one in ten thousand. This shows that fifteen minutes after being poked the eggs have established a block to polyspermy, one of the defining functional characteristics of activation. Hence, although the first visible indication of activation –the formation of the first cleavage furrow- will not be seen for six hours, we can conclude that the egg is undergoing the normal activation processes within minutes of being stimulated. Conclusions In an effort to conserve declining populations of animals, the development of protocols for the artificial manipulation of reproduction is of great interest. In the case of the neotropical frog E. coqui , we have observed that a large proportion of eggs that are laid unfertilized auto-activate. We showed that E. coqui eggs are easily activated by mechanical stimuli, leading to a need for careful manipulation of unfertilized eggs in all reproduction studies. Further, the cleavage pattern seen in mechanically activated eggs is similar to that of both auto-activated and chemically activated eggs, suggesting that mechanical stress, probably incurred in during oviposition, is responsible for the auto-activation mentioned above (table 1 ). Facile auto-activation of eggs has been reported in other species, complicating reproductive manipulation [ 48 ]. In E. coqui , this phenomenon may relate to internal fertilization, and it would be interesting to investigate auto-activation of unfertilized eggs in closely related, externally fertilizing species such as E. antillensis [ 38 ]. Some E. coqui eggs, however, remain intact and can be manipulated, showing signs of activation both at the morphological level, through the initiation of development, as well as the functional, through the slow block to polyspermy. By careful handling, we are now able to fertilize over half of the remaining, functionally viable eggs using a simple procedure. Finally, we have shown that E. coqui eggs do not degenerate rapidly and are capable of undergoing activation up to ten hours after deposition, thus creating a window of time for carrying out experimental procedures. Our results demonstrate efficient IVF in an internally fertilizing, terrestrial-breeding frog and help lay the foundation for future research and conservation possibilities in this unusually large genus of amphibians. Authors' contributions ET carried out the experiments and prepared the manuscript. SFM carried out preliminary experiments and supervised the manuscript. The study was conceived jointly. Both authors approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514616.xml |
516767 | Systemic endopolyploidy in Spathoglottis plicata (Orchidaceae) development | Background Endopolyploidy is developmentally regulated. Presence of endopolyploidy as a result of endoreduplication has been characterized in insects, mammals and plants. The family Orchidaceae is the largest among the flowering plants. Many of the members of the orchid family are commercially micropropagated. Very little has been done to characterize the ploidy variation in different tissues of the orchid plants during development. Results The DNA contents and ploidy level of nuclei extracted from various tissues of a tropical terrestrial orchid Spathoglottis plicata were examined by flow cytometry. Sepals, petals and ovary tissues were found to have only a 2C (C, DNA content of the unreplicated haploid chromosome complement) peak. Columns, floral pedicels of newly open flowers and growing flower stems were observed to have an endopolyploid 8C peak in addition to 2C and 4C peaks. In developing floral pedicels, four peaks were observed for 2C, 4C, 8C and 16C. In root tips, there were 2C, 4C and 8C peaks. But in the root tissues at the region with root hairs, only a 2C peak was observed. Nuclei extracted from young leaves shown three peaks for 2C, 4C and 8C. A similar pattern was found in the vegetative tissues of both greenhouse-grown plants and tissue-cultured plantlets. In mature leaves, a different pattern of ploidy level was found at different parts of the leaves. In the leaf tips and middle parts, there were 2C and 4C peaks. Only at the basal part of the leaves, there were three peaks for 2C, 4C and 8C. Conclusions Systemic variation of cellular endopolyploidy in different tissues during growth and development of Spathoglottis plicata from field-grown plants and in vitro cultures was identified. The implication of the findings was discussed. | Background In the classical cell cycle, the nuclear DNA contents vary only within the range of 2C and 4C, where C is the haploid DNA content per nucleus. When mitotic DNA replication in somatic cells is not followed by cell division (a process called endoreduplication), variation of cellular ploidy levels (designated as somatic polyploidy or endopolyploidy) can result [ 1 ]. Endopolyploidy is considered to be developmentally regulated [ 2 ] and has been described in several plant species including maize [ 3 , 4 ], sunflower [ 5 ], tomato [ 6 ], Arabidopsis [ 7 ] and brassicas [ 8 , 9 ]. Presence of endopolyploidy as a result of endoreduplication is also a common feature of insects and mammals [ 10 , 11 ]. In orchids, endoreduplication has been described in the raphid crystal idioblasts of Vanilla [ 12 ] and in parenchyma cells of Vanda seedlings [ 13 , 14 ]. The family Orchidaceae has an estimated 17,000 to 35,000 species, making it the largest and an important family of the flowering plants [ 15 ]. Many of the members of the orchid family are commercially valuable, and are micropropagated [ 16 ]. The explant sources used for orchid micropropagation include inflorescence, leaves, floral buds and roots [ 16 , 17 ]. However, very little is known about the ploidy variation in different explant tissues of the orchid plants during different developmental periods and at the stage when they are used as explants for micropropagation. Increased knowledge of the degree of endopolyploidy in the explant tissue source will be highly valuable for the maintenance of the original ploidy level in culture [ 8 ]. In this paper, systemic variation of cellular ploidy and DNA content in different tissues of Spathoglottis plicata , a common tropical terrestrial orchid species, from field-grown plants and in vitro cultures was investigated. Results Ploidy level of nuclei isolated from leaves and roots of greenhouse-grown plants Flow cytometry analysis of nuclear preparations from entire young leaves of 1–3 cm in length revealed that there were three peaks of fluorescence corresponding to 2C, 4C and 8C DNA content of somatic cells (Fig. 1A ). About 50% of the nuclei were found to have 2C DNA content, 25% were 4C and 15% were found to have 8C DNA content (Fig. 1G ). Figure 1 Nuclear DNA content and distribution of endopolyploid nuclei in vegetative tissues of the greenhouse-grown plants: A. Young leaves, B. Basal part of the mature leaves, C. Middle part of the mature leaves, D. Tip of the mature leaves, E. Root tips, F. Root segments with root hairs. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). G. The population of endopolyploid nuclei in tissues A-F. More detailed analysis was done on mature leaves of 48 cm in length. Tissues taken from different regions of the mature leaves showed that the pattern of ploidy levels was different at different regions. For tissues taken from the basal (petiolar end) of the leaves, there were three fluorescence peaks corresponding to 2C, 4C and 8C nuclear DNA content (Fig. 1B ). However, no 8C peak was observed from nuclei preparations taken from tissues of the middle (Fig. 1C ) and tip (Fig. 1D ) regions of the same leaf; only 2C (35–40% of cell populations) and 4C (60–65% of cell population) nuclei were identified (Figs. 1C,1D,1G ). In the young root tips, 2C, 4C and 8C peaks were observed (Fig. 1E ) and the distribution skewed toward 2C population, which accounted for more than 60% of the nuclei population analyzed (Fig. 1G ). The percentage of nuclei population with 4C and 8C DNA content in the root tip was relatively small and accounted for only about 10% each (Figs. 1E,1G ). Cells from root segments taken at least 2 cm away from the tips were all 2C (Fig. 1F ). Ploidy level of nuclei isolated from floral tissues Preparations from floral pedicels (Fig. 2A ), columns (Fig. 2B ) of freshly open flowers, and growing flower stems (Fig. 2F ) revealed that there were 2C, 4C and 8C nuclei. The proportions of 2C and 4C nuclei ranged from 40–50% (Fig. 2H ), and only about 8 – 10% of nuclei were found to be 8C (Fig. 2H ). In pedicels of un-open flower (Fig. 2G ), there was a 16C peak in addition to 2C, 4C and 8C peaks. The majority of nuclei were in 4C (28%) and 8C (46%) peaks. The 2C and 16C peaks each had less than 10% of the total nuclei (Fig. 2H ). Nuclei isolated from the sepals (Fig. 2C ), petals (Fig. 2D ) and ovary tissues (Fig. 2E ) were all 2C. Figure 2 Nuclear DNA content and distribution of endopolyploid nuclei in floral tissues of the greenhouse-grown plants: A. Pedicels, B. Columns, C. Sepals, D. Petals, E. Ovary tissues, F. Growing flower stems, G. pedicels of un-open flowers. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). H. The population of endopolyploid nuclei in tissues A-G. Ploidy level of cells from in vitro cultures Protocorms of S. plicata were found to have 2C, 4C and 8C nuclei (Fig. 3A ) with majority (over 70%) of them with 2C DNA content (Fig. 3E ). In the young leaves of plantlets, majority (70%) of the nuclei isolated were 4C, and about 20% of were 2C nuclei and the rest 8C (Figs. 3B,3E ). In the root tips of cultures, there were about 40% each of 2C and 8C nuclei, and the proportion of 4C nuclei was only about 10% (Figs. 3C,3E ). Nuclei taken from root tissues at the region with root hairs were all 2C (Figs. 3D,3E ). Figure 3 Nuclear DNA content and distribution of endopolyploid nuclei in vegetative tissues of the tissue-cultured plants: A. Protocorms, B. Young leaves, C. Root tips, D. Root segments with root hairs. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). E. The population of endopolyploid nuclei in tissues A-D. Discussion As references to the DNA content of gametic nucleus of individuals, DNA 'C' values have been estimated in several thousand animal and plant species [ 18 ]. For angiosperms, information on 'C' values is used in a wide range of biological fields [ 19 ]. The 1C DNA values in angiosperm plants differ approximately 1000 folds, ranging from 0.13 pg in Arabidopsis thaliana to 127.4 pg in Fritillaria assyriaca [ 18 ]. The DNA content per genome is usually considered to be constant between cells in an individual, and relatively constant between individuals of the same species [ 18 ]. However, in some plant species, intraplant ploidy variations were reported, and this implied that the nuclear DNA content in these species is not static and hence a great amount of variation occurs [ 8 ]. For example, a survey of Arabidopsis thaliana revealed endopolyploidy in hair trichomes, leaf epidermal cells, root tip cells, and cells in the hypocotyls [ 7 , 20 ], but not in the inflorescence [ 7 ]. In some cell types, the extent of endoreduplication appears to be intrinsically controlled by the differentiation programme, but environmental influences such as light can also affect endoreduplication [ 21 ]. The patterns of endopolyploidy may be affected by plant growth conditions in some plants. For example, leaves of in vitro grown tomato and potato plants were found to have lower level of endopolyploidy than leaves of plants grown in the greenhouse [ 22 , 23 ]. However, in S. plicata , patterns of endopolyploidy were found to be similar in both tissue-cultured plants and greenhouse-grown plants. Endoreduplication was found to occur in actively growing tissues with of S. plicata such as young leaves (1–3 cm in length, newly initiated) and root tips from greenhouse-grown plants. Similarly, endopolyploid cells were found in protocorms, young leaves and root tips from S. plicata seedlings in tissue culture. The common feature for protocorms, young leaves of 1–3 cm and root tips are that they are young and active in cell division and growth. In other orchids such as Dendrobium , endopolyploidy was found in root tips and newly expanded young leaves [ 24 ]. In the root segment with root hairs of S. plicata , endopolyploidy was neither found in tissue-cultured plants nor it was detected in greenhouse-grown plants. These results imply that the presence of the endopolyploidy during S. plicata development is an intrinsic programme, and it is not much affected by the growth condition. Besides root tips and newly developing young leaves, endopolyploidy was observed in mature leaves in a few Dendrobium species and cultivars [ 24 ]. Endopolyploidy was also detected in mature leaves of S. plicata. Furthermore, when the tip, middle and basal parts of the mature leaf were examined, different patterns of ploidy levels were obtained. Endopolyploidy was found only in leaf base part of mature leaves. A mature leaf represents a continuous developmental system, with the young, less green meristem cells at the basal petiolar end and the older, photosynthetically active cells at the tip [ 25 ]. Previous research shown that in cucumber and succulent plants with small genome, the level of endoreduplication does not increase once an organ is fully developed [ 26 , 27 ]. The tips and middle parts of mature leaves in S. plicata are fully developed. Endoreduplication in these tissues is unlikely since it would lead to further cell expansion. The pattern of DNA ploidy variation within the mature leaf is closely associated with the developmental status. The mechanism that resulted in endopolyploidy, however, remains unclear. In S. plicata , endopolyploidy was present in some floral tissues such as columns, growing flower stems and pedicels of both un-open and freshly open flowers. However, other floral tissues like sepals, petals and ovary tissues were found to have only 2C nuclei. In the growing un-open flower pedicels, the highest ploidy level even reached 16C. In cabbage, endopolyploidy was reported in cabbage flowers [ 8 ], and detailed patterns of endopolyploidy were found in various developmental stages of petals [ 9 ]. In cabbage petals, differentiation of expanding cells was characterized by endoreduplication [ 9 ]. In the proximal part of the cabbage petal, differentiation was accompanied with endoreduplication and cell enlargement. By contrast, no endopolyploid nucleus was found in the distal part of the lamina in the mature cabbage petal [ 9 ]. This study suggested that the developmental program of the cabbage petals might induce the initiation of endoreduplication [ 9 ]. In Arabidopsis , endopolyploidy was found in hypocotyls, cotyledonary leaves, rosette leaves, stems of bolting plants and floral leaves, but was not found in inflorescences [ 7 ]. Given the small size of columns within Arabidopsis floral buds, and the small population of endopolyploid nuclei found in columns and pedicels of S. plicata in this study, the minute population of the endopolyploid nuclei could easily be neglected when the whole floral buds were used for sampling. In S. plicata , it was found that the average size of nuclei was larger in columns and pedicels that have a measurable amount of endopolyploid cells than in other flora tissues without endopolyploidy (unpublished results). Similarly, a correlation was found between cell size and ploidy levels during cabbage petal development [ 9 ]. In Dendrobium , the post-pollination physiological changes were found to be different between floral tissues such as columns, ovary tissues, sepals and petals [ 28 ]. Edgar and Orr-Weaver [ 10 ] suggested that as endoreduplication is often found in large cells or cells with high metabolic activity, it might be a common strategy for cell growth without division. Further evidence was found in legumes where cell differentiation to a specialized function as pod wall tissues was accompanied by endoreduplication, and higher ploidy levels coincided with maximum pod growth [ 29 ]. During tomato fruit development, the pericarp tissue of young green fruit did not have higher ploidy (usually within 2C and 4C), but most of the cells in pericarp became endopolyploid (up to 256C) as the fruit developed further [ 6 ]. In tobacco single cell culture, endoreduplication was associated with plant growth regulators. When auxin was applied alone, endoreduplication was induced and the DNA content kept pace with the increment of cell volume. When both auxin and cytokinin were supplied subsequently, the cells divided first as amitosis leading to DNA endoreduplication, then followed by normal mitosis cell cycles [ 30 ]. Gibberellin and ethylene were found to play important roles in the endoreduplication of Arabidopsis hypocotyls [ 31 ]. In cabbage, mammals, Drosophila melanogaster and some small genome plants like Arabidopsis , it is thought that endoreduplication is developmentally regulated [ 8 , 10 , 32 ]. The systemic endopolyploidy revealed within different tissues of S. plicata raises the question of its possible implications. In tobacco, it was reported that the morphogenetic response of the tissues culture was related to the nuclear DNA content variation within stem explants of different ages [ 25 ]. In Oncidium Gower Ramsey, a hybrid orchid, only root tips, cut surfaces of stem segments and young leaves were able to form callus in tissue culture. Other explants such as old leaves and the roots without meristem tips could not form any callus [ 33 ]. Molecular data showed that the nuclear DNA modulation was closely related to the acquisition of embryogenic competence in cultured carrot hypocotyls [ 34 ]. In various tissues in cabbage plants, the number of endocycles was tissue-specific and was characteristic of the developmental stage [ 8 , 9 , 32 ]. These studies suggested that pattern of endopolyploidy may represent the characteristic of the developmental and physiological properties of the tissue. The role of endoreduplication in plant development is still not well understood. The presence of endopolyploidy was proposed to be associated with several factors, such as taxonomic position of a species, life cycle, genome size, and organ type [ 35 ]. Recent investigation of 16 plant species suggested that endopolyploidization might provide a mechanism to facilitate plant growth [ 35 ]. Endoreduplication benefits fast growth in several ways. In polyploid cells, the increased gene dosages may enhance the transcriptional and metabolic activities. In addition, several processes are eliminated in the endoreduplication cycle such as the reorganization of the cytoskeleton and condensation of the chromosomes, and that might allow faster growth [ 36 ]. Ploidy level also plays a role in controlling the size of the cells, the organs or the whole plant [ 6 , 9 , 37 ]. One of the common features of plant development is the uneven enlargement of plant cells coupled to somatic endoreduplication, which indicates that the enlargement of plant cells might be the consequence of the increased genome size [ 9 , 37 ]. This research may also have an impact on the orchid industry. Orchidaceae is the largest family of the flowering plants, and many of its members are commercially hybridized [ 38 ]. Clonal propagation is a common and essential practice for multiplication of hybrid orchids because the genotypes of the hybrids are usually heterozygous [ 39 ]. Many tissues have been used as explants for micropropagation including inflorescence, leaves, floral buds and roots [ 16 , 40 , 41 ]. Somaclonal variation is undesirable, and it is a major problem encountered in commercial micropropagation of orchids if true-to-type plants are required [ 39 , 42 ]. The mechanism of the somaclonal variation is poorly understood [ 42 ]. Polyploidy is considered as a possible cause for somaclonal variation in tissue cultures [ 43 ], but how polyploidy is generated during tissue culture is unclear [ 39 , 42 ]. The presence of systemic endopolyploidy and DNA content variation within different tissues of S. plicata as revealed in this study suggests that endopolyploidy and DNA content variation in explants might be a cause for somaclonal variation in tissue culture derived orchid plantlets. Thus, the pre-knowledge about the ploidy variation in different explant tissues is valuable for clonal propagation or for deliberate induction of variants in culture. Further systemic investigation of the relationship between somaclonal variation and type and endopolyploid level of source explants will provide indepth knowledge for micropropagation of orchids. Conclusions Systemic variation of cellular endopolyploidy in different tissues during growth and development of Spathoglottis plicata from field-grown plants and in vitro cultures was developmentally regulated. Pattern of endopolyploidy is a character of the developmental and physiological properties of the tissue. This finding provides useful information for understanding of the plant development and for industrial propagation of orchids. Methods Plant materials Spathoglottis plicata L. is a common tropical terrestrial orchid. The plants were grown in pots and placed in the greenhouse at 28 ± 4°C without artificial lighting. The following materials were taken for analysis: a) young leaves (1–3 cm in length), b) mature leaf (48 cm in length), c) root tips (2 cm including the tip), d) root segments from region with root hairs (2 cm away from the root tip), e) newly opened flowers, f) growing flower stems and g) developing floral pedicels of un-open flower (4–6 days before flowering). Seedpods were surface-sterilized for 20 min in 20% (v/v) Clorox™ solution and subsequently rinsed 3 times with autoclaved water. Seeds were germinated aseptically in 9 cm diameter petri dishes containing 25 ml Knudson C orchid medium (Duchefa, Netherlands) with 2% (w/v) sucrose and 0.8% (w/v) agar. All cultures were incubated at 25 ± 2°C under a 16 h photoperiod (light intensity: 54 μm -1 m -2 s -1 ). The following materials from in vitro cultures were examined: a) protocorms (6 weeks after germination), b) young leaves (1–2 cm in length) from seedlings, c) root tips (1 cm segments from tips), d) root segments with root hairs (at least 1 cm away from the root tips). Preparation of nuclei and flow cytometry analysis of nuclear DNA content Extraction of nuclei and staining of DNA were performed according to the method of Arumuganathan and Earle [ 44 , 45 ] with some modifications. All preparations were done on ice. Tissues (about 0.3 – 1.0 g) were sliced with razor blades into strips of less than 1 mm in 1 ml extraction solution (1 mM MgSO 4 , 5 mM KCl, 0.5 mM HEPES, 1 mg/ml dithiothreitol, 2.5 mg/ml Triton X-100, pH 8.0) and extracted for 45 minutes. After filtering through a 45 μm Falcon cell strainer, 100 μl of propidium iodide (1 mg/ml) and 2.5 μl of 500 μg/ml DNase-free RNase (Boehringer Mannheim, Indianapolis, IN) were added to each sample followed by 30 min incubation at 37°C. A Coulter EPICS ® Elite ESP with 15 mW 488 nm Cyonics Argon air-cooled laser flow cytometer was used to measure the relative fluorescence of nuclei. For each sample, at least 10,000 nuclei were analyzed. Data were analyzed with WinMDI27B software (Joseph Trotter™). Abbreviations C, DNA content of the unreplicated haploid chromosome complement PI, propidium iodide PMT4, photo multiplier tube Authors' contributions MY carried out experiments in the project. CSL and MY prepared the manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516767.xml |
548141 | Predominant constitutive CFTR conductance in small airways | Background The pathological hallmarks of chronic obstructive pulmonary disease (COPD) are inflammation of the small airways (bronchiolitis) and destruction of lung parenchyma (emphysema). These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter. Methods We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole. Results In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential (TEP; lumen to bath) of the bronchiole was small (mean ± sem: -3 ± 1 mV; n = 25), but when gluconate replaced luminal Cl - , the bionic Cl - diffusion potentials (-58 ± 3 mV; n = 25) were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl - permeability was at least 5 times greater than Na + permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM)+IBMX (100 μM), ATP (100 μM), or adenosine (100 μM), but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM), GlyH-101* (5–50 μM), and CFTR Inh -172* (5 μM). RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways. Conclusion These results indicate that the small airway of the pig is characterized by a constitutively active Cl - conductance that is most likely due to CFTR. | Background Most, if not all, forms of chronic obstruction pulmonary disease (COPD) as well as asthma begin in the small airways. While the pathogenesis of small airway diseases is poorly understood [ 1 , 2 ], it is generally accepted that the fluid and electrolyte transport properties of the epithelia lining these peripheral bronchioles play a crucial role in maintaining normal airway hygiene and patency. Some argue that these fluids are the primary defense because coupled with the ciliated escalator they form the first mechanism for clearing the airway of foreign debris and noxious agents. At the same time, almost nothing is known with certainty about the transport properties of distal airway epithelia or how fluid movements help maintain hygiene. No doubt, the paucity of understanding is due to the inaccessibility and the fragility of the tissue. Most concepts of the mechanisms and functions at this level have been taken from findings in the upper respiratory tract or from the larger cartilaginous ringed structures of the trachea and bronchi [ 3 - 7 ]. More extrapolations have been made from primary cultures of the same sources [ 8 , 9 ]. Two previously published attempts were made to measure electrolyte transport parameters in isolated segments of small airways dissected from the peripheral airways of sheep [ 10 - 12 ] and pigs [ 13 , 14 ]. However, in these studies the electrical signals, reflecting underlying transport properties may have been severely muted by tissue trauma during dissection and preparation. For standard electrophysiological studies of epithelia, dissection of the bronchiole would seem mandatory in order to maintain control of solutions on both sides of the epithelium. In order to minimize trauma, however, we attempted to microperfuse small bronchioles (i.d. 0.5–0.8 mm) in the periphery of pig lung without dissection. Unfortunately, since the bronchioles are embedded in a parenchyma of bronchioli and alveoli, this approach sacrifices control of the contra-luminal solution. Nonetheless, under this condition, we now find striking improvements in electrophysiological responses and strong evidence of a highly Cl - selective conductance that dominates the electroconductive properties of this epithelium, that is most probably duo to CFTR. Methods Tissue Lungs were excised intact immediately after sacrifice of young pigs (30–60 kg). Lungs were maintained inflated through a ligated plastic tube connected to an aquarium air pump (~1 L/min) to maintain a positive airway pressure of 10–14 cm-H 2 O. The assembly was wrapped in a plastic bag and transported from the abattoir to the laboratory (<60 min) in an insulated box chilled with ice. In the laboratory, small pieces of about 0.5 cm 3 were cut from the peripheral lung parenchyma, usually from along the costal diaphragmatic ridge of the lower lobes. In general, the freshest tissue gave the best responses although some tissue responded well after 6–8 hours of storage in a cooled environment. Microperfusion Under a dissecting microscope, the opening to a small airway was visualized on the proximal cut surface of a small block of lung tissue. The airway opening was then cannulated with a system of two concentric micropipettes [ 15 - 17 ] with tips fabricated so that the identified open end of the bronchiole could be aspirated into the outer pipette. (Fig. 1 ). Simultaneously, a double barreled inner pipette was inserted into the lumen of the bronchiole for delivering experimental solutions and monitoring electrical potentials through one barrel while constant current pulses were delivered through the other barrel [ 18 ]. Figure 1 Pipette assembly for microperfusing segments of undissected bronchiole. The bronchiole is held in outer large pipette (A) by suction. An inner, septated cannulating pipette provides current passing capacity through one barrel (B 1 ) and perfusing fluid to the duct lumen through the opposite barrel (B 2 ), which also contains a small cannula pipette (C) that allows changes of perfusing solutions. Solutions The perfused airways were intact and therefore remained embedded in the mass of connective tissue and air filled alveoli that normally surround the bronchi in vivo . The surrounding parenchymal tissue effectively prevented changing the solution in contact with the serosal surfaces of the airway epithelium, which in vivo is the extracellular fluid and in the intact preparation could not be readily removed. NaCl Ringer solution is designed to mimic mammalian extracellular fluid. Therefore, we used NaCl Ringer in the bath to establish electrical continuity with the serosal surface of the bronchiolar epithelium during the entire experimental period. The Ringer solution contained in (mM): Na + (~155), K + (4.5), Mg 2+ (1.2), Ca 2+ (1.0), PO 4 3- (3.5), Cl - (152), SO 4 2- (1.2), Glucose (5) buffered to pH 7.4 with NaOH. For ion diffusion studies, 150 mM of Cl - was replaced with an equivalent amount of gluconate (taken as impermeant), HCO 3 - , NO 3 - , I - , or Br - . Luminal solutions perfusing the bronchiolar airway were rapidly changed as needed via a manifold distributing stores of the above solutions through a needle tube to the tip of the perfusing pipette (Fig. 1 ). Agonists were added to solutions (in μM) as needed as forskolin (1), IBMX (100), ionomycin (1), ATP (100), and adenosine (100). Inhibitors were added (in μM) as needed as amiloride (10), NPPB (100), CFTR Inh -172 (5) [ 19 , 20 ], GlyH-101 (50) [ 21 ] (CFTR Inh -172 and GlyH-101 were generous gifts from Dr. A. Verkman, University of California, San Francisco, CA.). Electrical Measurements The basic electrical circuit for recording potentials and conductance during microperfusion has been described previously [ 18 ]. The lumen of the bronchiole can be considered as a conductive core of fluid (perfusate) surrounded by an insulating epithelium.Unfortunately, the complex arborizing geometry of the bronchiole make it impossible to calculate the specific conductance of the epithelium from cable analysis as is possible with straight, unbranching tubes like sweat ducts and renal tubules. Thus, in the present protocol, the current pulse induced voltage deflections reflect the total resistance of the preparation and can only be used to compare changes in the epithelial resistance within the same preparation when identical solutions are present in the lumen and bath; e.g., pre- and post-drug application. Although we recorded the total resistance (R t ) of the system, which includes the summed resistances of the epithelium (including parallel shunts through it) plus the core resistance of the lumen plus the extracellular fluid resistance, the resistance of the epithelium relative to R t was small (even after floating the current passing circuit), and therefore changes in the epithelial resistance were obscured. Consequently, the response of TEP's was taken as the primary indication of the permeability properties of the epithelium. Temperature The bathing solution was maintained at 35 ± 2°C. mRNA expression Total RNA was isolated from the dissected bronchioles of 4 pigs by using RNeasy Mini Kit (QIAGEN Inc. CA). RNA was reversely transcribed using Sensiscript RT Kit (QIAGEN Inc. CA). The resulting first-strand cDNA was directly used for PCR amplification (TaqPCR Core Kit, QIAGEN Inc. CA). The conditions for PCR reactions were as follows: 3 min at 94°C (initial melt); 35 cycles of 1 min at 94°C, 1 min at 55–60°C, 1 min at 72°C and then 72°C 10 min (final extension). For the negative control, RT-PCR was performed in the absence of RT. The PCR products were analyzed by agarose gel electrophoresis stained with ethidium bromide. The primers were constructed on the basis of the published cDNA sequence of CFTR, ENaC, NKCC1 and β-Actin from GenBank. Since the pig gene sequence was not complete, primers were obtained from the human accordant gene, in which highly conserved regions were selected. The pairs of primers for CFTR (accession no. NM_000492) were sense 5'-TCCTAAGCCATGGCCACAA-3' and antisense 5'-GCATTCCAGCATTGCTTCTA-3'; sense 5'-GCCTGGCACCATTAAAGAAA-3' and antisense 5'-CTTGCTCGTTGACCTCCACT-3', which generated a 197-bp and 171-bp CFTR PCR product respectively; for α-ENaC (Z92978) were sense 5'-CAACAACACCACCATCCAC-3' and antisense 5'-TAGGGATTGAGGGTGCAGA-3', which generated a 225-bp PCR product; for β-ENaC (NM_000336) were sense 5'-TGCTGTGCCTCATCGAGTTTG-3' and antisense 5'-TGCAGACGCAGGGAGTCATAGTTG-3', which generated a 277-bp PCR product; for γ-ENaC (X87160) were sense 5'-TCAAGAAGAATCTGCCCGTGA-3' and antisense 5'-GGAAGTGGACTTTGATGGAAACTG-3', which generated a 237-bp PCR product; for NKCC1 (U30246) were sense 5'-TCCAGGTAATGAGTATGGTGTCAG-3' and antisense, 5'-GTTAAGATGTAGCCACGAAGAGGT-3', which generated a 205-bp PCR product; and for β-Actin (BC004251) were sense, 5'-TTCAACTCCATCATGAAGAAGTGTGACGTG-3' and antisense, 5'-CTAAGTCATAGTCCGCCTAGAAGCATT-3', which generated a 312-bp PCR product. All primers showed products closely corresponding to the predicted size for expression of RNA transcripts for these genes. Immunocytochemistry The bronchioles were dissected and then fixed in ice-cold 4% formaldehyde buffered in phosphate at 4°C for 3 hours, infiltrated with cryoprotectant (30% sucrose in PBS) overnight, and frozen in OTC medium (Triangle Biomedical Sciences) at -35°C. Sections of 5 μm thickness were cut on a cryostat microtome (Thermo Electron) and mounted on glass slides (Fisher Superfrost Plus). Antigen retrieval was performed using a pressure cooker (10 min in 10 mM citrate buffer, pH 6). To reduce autofluorescence, sections were treated for 20 min with 1.5% sodium borohydride in PBS. Sections were incubated sequentially with blocking solution (30 min), primary antibody (overnight at 4°C), and secondary antibodies conjugated to Alexa Fluor-488 and/or -546 (Molecular Probes). Confocal images were acquired with a Zeiss LSM-510 microscope and assembled using Adobe Photoshop. CFTR was labeled with rabbit antibody R3194 (courtesy of C. Marino), and ENaC with a rabbit antibody against the β-subunit of human ENaC (kindly courtesy of C. Fuller and D. Benos). Tight junctions were labeled with a mouse antibody against the junction-associated protein zonula occludens-1 (Zymed). Nuclei were stained with TO-PRO-3 (Molecular Probes). Statistical treatment Differences in mean measurement were assayed by applying the Student T test to paired or unpaired means as appropriate. A probability (P value) of ≤ 0.05 was taken as significantly different. Results Basic electrical properties When the bronchiolar lumen was perfused with NaCl Ringers, which we assumed represented insignificant ion gradients except for a small lumen (145 mM) to serosa (110 mm) Cl - gradient. Despite the fact that this small Cl - gradient should render the lumen positive, there was a small spontaneous lumen negative potential of about -3 mV (Fig. 2 ; Table 1 ). When we applied amiloride (10 μM) to the lumen to block Na + conductance (gNa + ), the TEP decreased slightly, but without statistical significance (Fig. 2 ; Table 1 ). We were unable to detect a change in total conductance with amiloride applied to the lumen. Figure 2 Effect of amiloride and Forskolin (Fsk, 5 μM) + IBMX (100 μM) on transepithelial potential (TEP) of bronchiole. In the presence of luminal Cl - , the effects of both amiloride and Fsk+IBMX on TEP were almost imperceptible (left side). However, when Cl - was substituted with Gluconate to more effectively reveal the Cl - conductance, addition of amiloride depolarized TEP and Fsk+IBMX hyperpolarized the TEP (right side), suggesting that the large Cl - conductance present in the epithelium mutes (shunts) the smaller changes in conductance occasioned by amiloride and Fsk when isotonic Cl - is present bilaterally. NaGlu: Na-gluconate Table 1 Amiloride Inhibition NaCl NaCl+Amil NaGlu NaGlu+Amil TEP (mV) -3.1 ± 0.6 -2.6 ± 0.7 -57.3 ± 2.7 -43.6 ± 2.7 Δ TEP (mV) -- +0.5 -- +13.7 n 25 10 25 16 P value -- 0.676 -- <0.001 Gluconate Substitution However, when we replaced luminal Cl - with the impermeant anion, gluconate, the TEP hyperpolarized to as much as -90 mV (Fig. 3 , Table 1 ). Addition of amiloride (10 μM) to the lumen depolarized the mean TEP of these tissues by an average of 14 mV (Fig. 2 ; Table 1 ). Figure 3 Effect of luminal Cl - substitution in a perfused small bronchiole on TEP. The substitution of Gluconate, an impermeant anion, for permeable Cl - in the lumen markedly hyperpolarized the TEP. This response indicates a predominant Cl - conductance that appears to be constitutively active. Anion Conductance Inhibitors Under conditions of symmetrical [Cl - ] concentrations, luminal applications of anion conductance inhibitors had virtually no detectable effect on the spontaneous TEP. However, under hyperpolarizing conditions created by Cl - substitution in the lumen, NPPB (100 μM), GlyH-101 (50 μM) and CFTR Inh -172 (5 μM) significantly depolarized the TEP by 36.7, 20.0 and 8.7 mV (Fig. 4 ; Table 2 ). Bumetanide (1 mM) had no effect on the TEP in either the presence or absence of a Cl - gradient (not shown). Figure 4 Effect of Cl - conductance inhibitors on TEP. In the absence of luminal Cl - (Gluconate substitution), two new inhibitors GlyH-101 (50 μM) and CFTR Inh -172 (5 μM, re: A. Verkman) showed at least partial inhibition of the Cl - :Gluconate anion diffusion potential. NPPB (100 μM) seemed to be the most effective inhibitor in terms of depolarizing the TEP. Table 2 Cl - Conductance Inhibition CFTR inh -172 NPPB GlyH-101 TEP (mV) -48.6 ± 4.7 -16.5 ± 2.1 -26.6 ± 2.5 Δ TEP (mV) +8.7 +36.7 +20.0 n 3 5 4 P value 0.03 0.001 0.03 Anion selectivity We assayed for the relative permeability of several monovalent anions by substituting them for Cl - in NaCl Ringer. Amiloride (10 μM) was added in order to block Na + transport. For luminal Cl - , Br - , I - , NO 3 - , HCO 3 - and gluconate, the mean estimated P x /P Cl were, respectively, 1.0, 0.92, 0.79, 0.65, 0.33 and 0.28 (Table. 3 ). When the NaCl Ringer perfusion solution was diluted by 1:2, the potential depolarized by 12 ± 1 mV, indicating Cl - permeability exceeded the Na + permeability by about 5.4 fold (Fig. 5 ). Table 3 Anion selectivity sequence of the perfused bronchiole. The sequence of the bronchiole (upper data) roughly fits the known sequence of CTFR in other tissues (lower data taken from literature; cf. text) Airway Anion selectivity: Cl - ≈ Br - > I - > NO 3 - > HCO 3 - >> Gluconate Transepithelial TEP : ~0 -1.9 -5.1 -9.2 -19.5 -44.8 Estimated P x /P Cl : 1.0 0.92 0.79 0.65 0.33 0.28 CFTR anion selectivity: NO 3 - ≈ Br - ≈ Cl - > I - > HCO 3 - >> Gluconate Estimated P x /P Cl : 1.1 1.1 1.0 0.39 0.014 ~0 Figure 5 NaCl dilution diffusion across the bronchiolar epithelium on TEP. The hyperpolarization of the TEP with diluted NaCl (75 mM) indicates that Cl - must be significantly more permeable through the epithelium than Na + . Agonists When we added forskolin (5 μM) plus IBMX (100 μM), adenosine (100 μM), ATP (100 μM) or ATP (100 μM)+adenosine (100 μM) to the perfusate to activate CFTR gCl - in the presence of isotonic Cl - concentrations and in the absence of a hyperpolarizing gradient, the TEP did not change perceptibly (not shown), but in the presence of the Cl - gradient, the TEP hyperpolarized significantly to all agonists; the response appeared to be increased when both ATP and adenosine were added together (Fig. 6 ; Table 4 ). In order to observe the maximum effect on the activation of Cl - conductance, amiloride (10 μM) was present in all luminal perfusates to block ENaC Na + conductance. Figure 6 Effect of Cl - conductance agonists on TEP: Applying Fsk (5 μM)+IBMX (100 μM), adenosine (100 μM), ATP (100 μM) and ATP (100 μM)+adenosine (100 μM) to the perfusate to activate CFTR gCl - in the presence of a hyperpolarizing Cl - gradient, the TEP hyperpolarized significantly; however, the response appeared to be increased when both ATP and adenosine were added together. In order to observe maximum effect on activation of Cl - conductance, amiloride (10 μM) is present in all luminal perfusate above to block Na + conductance. Table 4 Cl - Conductance Agonists Fsk+IBMX ATP Adenosine ATP+Adenosine Ionomycin TEP (mV) 53.0 ± 2.8 -51.0 ± 0.8 -55.8 ± 6.7 -52.0 ± 6.5 -40.1 ± 7.5 Δ TEP (mV) -26.0 -5.5 -5.5 -6.3 0.4 n 13 4 5 6 5 P value <0.001 0.007 0.003 0.011 0.836 RT-PCR Two sets of different primers for CFTR as well as primers for α-, β-and γ-ENaC, NKCC1, and β-Actin all produced products of predicted sizes for each of the corresponding mRNAs (Fig. 7 ). Figure 7 RT-PCR bands for CFTR (lanes 2 &3), α,β,γ subunits of ENaC (lanes 5,6,7 respectively), and NKCC1 (lane 9) and β-Actin (lane 10). Two independent sets of primers were used to detect CFTR. The presence of CFTR, ENaC, and NKCC1 may indicate that the epithelium has both absorptive and secretory functions. β-Actin was used as a housekeeper marker for control. Size ladders in 100 bp increments with lowest band equal to 100 bp (lanes 1,4,8). Immunocytochemistry of CFTR Immunoreactive CFTR antibody (gift of C. Marino) was detected in the apical domains of the bronchiolar epithelia in a continuous border of the bronchiole. The tight junctions were labeled simultaneously and appeared as punctate areas within the border staining for CFTR consistent with the expected location for these structures (Fig. 8A and inset). Negative controls consisted of omission of primary antibodies and showed no labeling of the antibody in any tissue (Fig. 8B ). Figure 8 Immunocytochemical localization of CFTR in bronchiole epithelium. A.). Antibody R3194 against CFTR prominently labels (green) the apical margin of epithelial cells lining the bronchiole cut in cross section. Inset: high magnification view of epithelial cells showing demarcation of apical margin by tight junction-associated protein ZO-1 (red). B.) Control serial section stained without primary antibody. Discussion The airways are extraordinarily specialized conduits for air to and from the alveoli for gas exchange. They must remain moist in order to remain flexible and to effectively filter air before it enters the delicate tissues of the alveoli. Hence, a principal function of the airway epithelia is to provide and service a continuous layer of aqueous fluid on the airway surfaces. For decades, the upper airways, trachea, and large bronchi have served as models for the entire tracheobronchial epithelial function [ 14 , 22 , 23 ], and yet it is known that there are distinct regional differences progressing from nares to alveoli. For example, anatomically, the upper airways and bronchi are characterized by submucosal glands that secrete fluid directly into the airway, but are absent in the peripheral small airways [ 24 ]; [ 25 ]. Likewise, the trachea and larger airways are kept patent by cartilaginous rings whereas the peripheral airways are generally held open (but may collapse) by internal retractile forces of the lung parenchyma [ 26 ]. Similarly, the cell populations change. The epithelium of the upper respiratory tract changes from ciliated, pseudostratified columnar to simple cuboidal cells in the smaller airways where the proportion of Clara cells increase and that of ciliated cells decrease [ 27 ]. Functionally, there are differences as well. For example, in humans and in dogs, the spontaneous transmural electrical potential appears to become less negative (lumen: blood) from upper to lower airways [ 13 , 28 , 29 ]. Differences in airway surface fluid composition may also exist [ 30 ]. The functions of the lower peripheral airway epithelium is poorly defined and understood because this tissue is inaccessible and relatively unamenable to standard physiological techniques for study. Nonetheless, a few courageous attempts to unravel these mysteries have been made. About ten years ago, Ballard [ 13 , 14 ] and Al-Bazzaz [ 10 ] isolated and perfused small airways and bronchioles from pig and sheep, respectively. Ballard perfused porcine airways of about 1 mm diameter and reported a mean spontaneous TEP potential difference in bilateral NaCl Ringer solution of about -3.4 mV and in lumen Cl - free solution of about -16 mV [ 14 ]. Al-Bazzaz perfused ovine bronchioles of about 250 μm diameter and reported a mean spontaneous TEP of -2.5 mV in bilateral NaCl Ringer and of about -4.2 mV mean TEP in lumens perfused with Cl - free Ringers [ 11 ]. In all of these studies, it has been difficult to ascertain to what degree the electrical responses were muted or altered by trauma to the tissue during dissection because similar measurements are not possible in vivo . We, too, found similar, relatively small TEP voltages in microdissected porcine airways. Undissected bronchioles Therefore, in order to maximally preserve, and minimally traumatize the airway epithelium, we avoided dissecting the bronchial structure and microperfused the airways imbedded in lung parenchyma. We found that the spontaneous TEP in bilateral Ringer solution was about -3 mV, slightly more negative than reported earlier. But in striking contrast to the previous studies (including our own dissected preparations), we found that the bi-ionic TEP with Cl - free solutions in the lumen was as much as -90 mV (mean: -57 mV; Table 1 ; Fig. 2 ). These differences almost certainly reflect differences in trauma to the airway. Because of the extremely complicated morphology that arises from arborization of the airway in route to hundreds of alveolar acini, it is impossible to physically remove the surrounding tissues (very small bronchioles, respiratory bronchioles, blood vessels, and alveolar sacs) without breaking or tearing smaller "branches" from the "tree" of airways. Both Ballard and Al-Bazzaz attempted to patch these breaks by micro-suturing obviously dangling limbs; unfortunately, only larger branches are amenable to such heroic attempts and many smaller, even microscopic transepithelial openings, must remain. In order for an epithelium to reflect its in vivo electrical properties in vitro , it is fundamental that the integrity of the epithelial sheet be conserved because TEP measurements depend on separation of charge. Breaks, holes, or tears in the barrier inescapably create electrical shunts, which, by allowing simultaneous back leak of epithelial current, prevent separation of charge. Even though the individual cells or groups of cells of the epithelia function and respond physiologically, the transepithelial voltage signals will be erroneously muted or lost through such shunts. In the present case, it appears that perfusion of the bronchiole without dissection from surrounding parenchyma, minimizes trauma induced shunts and allows detection of a much more complete and robust electrical signal. Unfortunately, the price for this preservation of signal is the inability to control the contra luminal solution. Keeping in mind that the undissected bronchiole is embedded in a mass of air filled alveoli together with numerous other elements of the tissue at this level, it is easy to see that changing the bath solution can have no acute effects on the composition of the contra luminal solution at the basilateral membrane of the epithelium. Consequently, we did not change the bath solution and assumed that normal Ringer solution is sufficiently similar to extracellular fluid in vivo to avoid introducing significant free solution diffusion potentials or altering the physiological composition of the native fluid present in the extracellular compartment of the in tact preparation. Cl - Conductance The simple fact that substituting Cl - in the lumen spontaneously created a large lumen negative transmural potential (Figs. 2 , 3 ; Table 1 ) immediately indicates that the small airway is characterized by a dominant constitutively active anion selective conductance. The fact that imposing a putative 1:2 NaCl diffusion gradient across the epithelium resulted in -12 mV hyperpolarization indicates that the intact bronchiole epithelium is at least 5 times more permeable to Cl - than to Na + (Fig. 5 ). The facts that this Cl - conductance is immediately evident upon initial perfusion without any prior agonist stimulation and that additions of agonists did not significantly hyperpolarize the bronchiole perfused with 150 mM NaCl and hyperpolarized bronchioles perfused with Na Gluconate only by about 20–25% demonstrates that the Cl - conductance is constitutively open under these conditions (Fig. 6 ). That is, from the first moment of measurement after cannulation, the large Cl - conductance is present (Figs. 2 , 3 , 4 and 5 ). There was no need to activate PKA or wait for the transmural potential to develop even though the addition of forskolin, adenosine, and ATP appeared to increase Cl - conductance (Figs. 2 , 6 ; Table 4 ). In secretory cells where secretory activity is usually an acute, temporal event, CFTR is assumed to remain closed until activated by PKA and ATP. However, in the human sweat duct, also a rich source of CFTR, where the transport function is exclusively absorptive and where CFTR is thought to be the only anion conductance through the tissue, CFTR appears with a large Cl - conductance at the first moment of perfusion and measurement, indicating a constitutively open state for the CFTR channel in this tissue as well. It is well established that CFTR can be regulated in the classic sense by PKA phosphorylation and ATP in the sweat duct, but the cytoplasm must first be "rinsed" of small solutes by permeabilizing the basilateral membrane [ 31 ]. CFTR Since obstructive airway disease in Cystic Fibrosis arises in the small airways [ 32 - 34 ] and CF is known to be due to mutations in the CFTR gene that expresses a Cl - channel, we asked if this conductance could be due to CFTR. We found several lines of evidence that are consistent with CFTR being responsible for the Cl - conductance. First, the anion selectivity sequence, excepting NO 3 - , is grossly the same as that for CFTR (Table 3 ); i.e., Cl - ≈ Br - > I - > NO 3 - > HCO 3 - > Gluconate, and compares favorably with that of CFTR: SCN > NO 3 - > Br - > Cl - > I - > HCO 3 - > F - > ClO 4 - > gluconate [ 35 - 37 ]. Second, it is well known that CFTR is activated characteristically by cAMP mediated protein kinase A, which is driven pharmacologically by forskolin and IBMX. Here, we see (Figs. 2 , 6 ; Table 4 ) that these agonists routinely elevate the bionic Cl: gluconate diffusion potential by an average of -22 mV (n = 13). Similarly, ATP and adenosine may activate CFTR in airway epithelia since these agonists can elevate cAMP via purinergic receptors. [ 38 - 40 ]. In contrast, when we applied ionomycin to elevate intracellular Ca 2+ , there was no effect (not shown), suggesting that since CFTR is not sensitive to Ca 2+ mediated activation either, a.) the conductance is due to CFTR, b.) other Ca 2+ activated Cl - conductances must be fully constitutively activated or not present, or c.) luminal application of the ionophore drug does not increase intracellular Ca 2+ effectively. Third, CFTR is known to be inhibited by NPPB, CFTR Inh -172, and GlyH-101[ 19 , 21 ]. These inhibitors had varying, but consistently inhibitory effects on the TEP (Fig. 4 ; Table 2 ) indicating inhibition of Cl - conductance in the airway epithelium. However, the fact that none of the inhibitors appeared to completely inhibit the transepithelial potential might argue that another anion channel conductance is present. Two points diminish this argument. First, if the tissue is actively transporting, anion channel inhibitors will not completely ablate the TEP because that component of potential generated by the basilateral K + emf and the apical Na + emf should not be blocked and should be reflected across the epithelium in the TEP. Secondly, even when the CFTR Cl - channel has been isolated from active transport components, anion channel blockers do not usually completely block its conductance in native tissue [ 41 ]. Moreover, recently, in contrast, to our results, GlyH-101 was reported to be ineffective in blocking the Cl - conductance of pig nasal airways [ 42 ]. It is not known whether CFTR is present in the nasal epithelium of pigs, but biochemically, we found markers for conserved regions of expressed CFTR RNA from reverse transcriptase polymerase chain reactions (Fig. 7 ) in bronchioles dissected free of parenchyma post perfusion. Appropriate bands for CFTR protein in lysates of frozen peripheral lung tissue were also observed with affinity purified antibodies (J. Riordan, personal communication), and as shown in (Fig. 8 ) CFTR localized immunocytochemically, specifically to the apical surface of the bronchiolar epithelia. These data strongly suggest that CFTR is a part of, or probably is, the primary source of the anion conductance in small airways. Transport Function Not only does the bronchiole resemble the sweat duct with respect to exhibiting a constitutively active Cl - channel that responds to activation of PKA, but both are also apparently insensitive to ionomycin. They both show equally large bionic diffusion potentials for Cl - and are several fold more permeable to Cl - than to Na + , and yet both have very low transmembrane potentials in bilateral isotonic Ringer solutions [ 43 ]. Both are incompletely inhibited by anion channel blockers [ 20 , 41 , 44 ], and both are sensitive to amiloride. On the basis of these observations and by analogy with the sweat duct, it is tempting to propose that the bronchiole at least in its basal state is a constitutively absorptive epithelium. Conclusions We have found that the epithelium of terminal airways of the pig appears to express an anion permeability that constitutively dominates the electroconductive properties of this zone of the airway. Its anion selectivity sequence is similar to that expected for CFTR, and its activity can be enhanced by forskolin/IBMX or decreased by anion channel blockers known to inhibit CFTR. RT-PCR amplification products and specific antibodies identify CFTR in this tissue. The small airway appears to share a number of properties with the human sweat duct and may, by analogy, belong to a class of highly absorptive epithelia. Abbreviations CFTR: Cystic Fibrosis Transmembrane Conductance Regulator ENaC: Epithelial Na + Channel NKCC: Na + -K + -2Cl - Cotransporter TEP: Transepithelial Potential PKA: Protein Kinase A | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548141.xml |
340961 | Diverse Signals Establish the Left-Right Body Axis | null | Most animals (including humans) show a high level of bilateral symmetry: on the surface, the right side of our body resembles the left. A closer and deeper look, however, reveals an underlying asymmetry. The heart, for example, is on the left side in most humans, and the liver on the right. This left-right asymmetry develops early on in the embryo, and research in the past few years has revealed some of the molecular and cellular mechanisms that establish the left-right axis, which conveys positional information to cells in the growing embryo. We know that the formation of the axis relies on “crosstalk” between cells, which involves long-range signaling molecules (or ligands) and cell-surface receptors on cells that receive the signal. The molecules involved in the formation of the left-right axis during embryogenesis, along with their functions, are conserved among vertebrates. They include members of the Transforming Growth Factor beta (TGF-ß) family—such as the agonists (or ligands) Nodal, Vg1/GDF, and activin, and the antagonist (a molecule that interferes with agonist/ligands) Lefty—on the signaling side and members of the EGF-CFC family—such as the activin receptor and its coreceptors—on the receiving side. The EGF-CFC proteins play important roles in early vertebrate embryogenesis; mutations in these genes in the zebrafish (and mouse) result in a range of developmental defects, including problems in left-right axis specification. While ligand-stimulation of the activin receptor by Nodal and Vg1/GDF requires the EGF-CFC coreceptors, activin can activate the activin pathway without a coreceptor. Lefty—being an antagonist—can block activation of the activin receptor, though it is not clear how. Through genetic and biochemical studies in zebrafish and frog embryos, Simon Cheng, Alex Schier, and colleagues have now clarified a piece of this very complex signaling puzzle by demonstrating that Lefty inhibits a subset of TGF-ß signals—Nodal and Vg1/GDF but not activin—by blocking EGF-CFC coreceptors. They went on to show that a short, specific region of the signal molecules—accounting for less than 4% of the entire protein—determines whether the signals activate the activin receptor in an EGF-CFC coreceptor-dependent or -independent fashion and therefore governs susceptibility to Lefty. These findings suggest that subtle sequence differences between related signals can dramatically influence their function. Gene families are thought to arise from gene duplications, and the studies described here illustrate how members of the same gene families can gain diverse roles by specific interactions with coreceptors and antagonists. Additional studies will be necessary to reveal the structural basis for the observed diversity. Zebrafish embryo heart loops correctly in wild-type, incorrrectly in mutant | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC340961.xml |
516798 | The Conservation Business | Direct payments to local communities to conserve wildlife could prove effective but is biodiversity a commodity that can be bought and sold? | The language of conservation is changing: protecting biodiversity is no longer just about ethics and aesthetics; the latest buzzwords are commodities and consumers. Traditionally, conservation initiatives have talked up the benefits they will bring to the global community—saving species, habitats, ecosystems, and ultimately the planet. But conservation also has its costs, and these are usually borne by local people prevented from exploiting the resources around them in other ways. It is unfair to expect a localised minority to pick up costs that ultimately benefit a dispersed majority, argue conservation biologists. There has to be more money made available by concerned individuals, non-governmental organisations, national governments, and international bodies, and there need to be better ways to spend this money if conservation is to be effective, they say. Biodiversity is a commodity that can be bought and sold. We are consumers and must pay. Costs and Benefits Kenya boasts one of the world's most spectacular networks of national parks and reserves covering around 60,000 km 2 of the country ( Figure 1 ). But devoting such a vast area to conservation has its drawbacks. It has been estimated that were this land developed it would be worth around $270 million to the Kenyan people every year. Similarly, two national parks in Madagascar are estimated to have reduced the annual income of local villagers by around 10%. Of course, protected areas do bring some benefits to neighbouring communities, most notably through tourism. But in many cases the rewards are not great, they are rarely distributed evenly among individuals, and do not necessarily outweigh the costs. Figure 1 The Masai Mara National Park in Kenya Courtesy of Charlotte Stirling. ‘The costs of conservation fall disproportionately on local people, whereas the benefits are dispersed,’ says Andrew Balmford, a conservation biologist at the University of Cambridge in the United Kingdom. National and global communities stand to benefit from conservation of tropical biodiversity, but they must pay if they want to realise that benefit, he says. Conservation expenditure in the developed world is only about a third of what is needed for effective protection of 15% of the earth's terrestrial habitats, an area just large enough to preserve a representative sample of species, habitats, and ecosystems in the medium to long term ( Balmford et al. 2003 ). The developed world must make up this funding shortfall, argues Balmford. What's more, there need to be smarter ways to spend the money that's available, he says. Conservation by Distraction In recent years, many funding bodies have taken an indirect approach to conservation, investing in projects that encourage people to take up alternative practices that are compatible with conservation rather than investing in conservation itself. Perhaps the best example of this ‘conservation by distraction’ is ploughing money into community-based ecotourism projects. Such initiatives aim to bring the benefits of tourism to local people, thereby encouraging them to preserve the biodiversity they have. It's an attractive idea. In the mid 1990s, the United States Agency for International Development was investing more than $2 billion a year in 105 conservation projects with an ecotourism component. Similarly, between 1988 and 2003, the World Bank funded 55 development projects that supported protected areas in Africa, 32 of which placed an emphasis on ecotourism. However, an absence of quantitative data and analysis has made it hard to judge whether these projects actually achieve their dual purpose of preserving biodiversity and simultaneously reducing rural poverty. ‘Much of the information about community-based ecotourism is anecdotal and subjective,’ says Agnes Kiss of the Environment and Social Development Unit at the World Bank. The real contribution of these initiatives to biodiversity conservation is debatable, she says. ‘Many community-based ecotourism projects cited as success stories actually involve little change in existing local land- and resource-use practices, provide only modest supplement to local livelihoods, and remain dependent on external support for long periods, if not indefinitely’ ( Kiss 2004 ). For example, communities involved in the Infierno Community Ecotourism Project in Peru have received nearly $120,000 from their share in a tourist lodge and wages for providing services to visitors. This may have increased the income for a minority that are lodge employees, but only one family, whose adult members were all employed by the lodge, could afford to live solely on tourism. In the community as a whole, the average annual income from tourism was only $735 compared with nearly $2,000 earned elsewhere. Most of the community was still heavily dependent on other activities, and most of those activities are somewhat disruptive of conservation goals, says Kiss. Johan du Toit of the Mammal Research Institute at the University of Pretoria in South Africa is also critical of this kind of indirect approach to conservation. At the heart of the argument for community-based ecotourism is the idea of the ‘ecologically noble savage’, he says—the notion that those living closest to nature will know what's best for it. ‘It's a wonderful idea, but it just doesn't work. Nowhere in the history of evolution has sustainability ever been naturally selected for,’ says du Toit. ‘The AK47 automatic assault rifle has replaced the bow and arrow.…Every individual in a rural community that's out hunting will shoot what he sees when he sees it, because if he doesn't somebody else will.’ Nowhere is this problem more evident than in the ecotourist paradise of the Galápagos Islands ( Figure 2 ), where a small minority of fishermen is coming into conflict with conservation aims with increasing regularity ( Box 1 ). ‘Things are going down very quickly,’ says one Galápagos guide. ‘The iceberg is starting to tip over, and we are going to lose everything.’ If it still pays locals to exploit the environment rather than take part in one of the world's most buoyant ecotourism industries, it is clear that ecotourism alone cannot solve the world's conservation problems. Many think that ‘direct payment’ could be a useful tool. ‘Direct payment, very boldly speaking, is paying people in rural areas not to bugger up their environment,’ says du Toit. ‘It's just like if we want exclusive artworks to be looked after in the Louvre Gallery in Paris. Somebody's got to pay for it,’ he says. ‘You can't expect the Parisians who live in that arrondissement to cover the costs.’ Figure 2 Ecotourist Paradise in the Galápagos Courtesy of Catriona MacCallum. You Get What You Pay for—You Should Pay for What You Want to Get For people living in developing countries, where most of the world's biodiversity exists, the short-term rewards of exploiting these natural resources are significant. Replacing indirect conservation measures, such as community-based ecotourism, with payments directly into the pockets of local people could turn out to be a much more effective way to stem this exploitation, argues Paul Ferraro, an economist at Georgia State University in Atlanta ( Ferraro and Kiss 2002 ). It could also bring far greater development benefits than indirect financial support, he says ( Box 2 ). An additional spin-off is that direct payments force conservation biologists to quantify and hence clarify their objectives, says John Hough, principal technical advisor on biodiversity for the United Nations Development Programme. ‘We know what we don't want,’ he says, ‘but we're not very good at saying what we do want.’ A hypothetical model simulating how Madagascar should distribute an annual conservation budget of $4 million reveals that direct payments would have protected some 80% of original forest compared with only 12% protected through a system of indirect incentives. What's more, the annual income of rural residents would have been twice that generated through indirect investment ( Conrad and Ferraro 2001 ). For Ferraro, the logic of direct payment is simple. He draws an analogy with a car journey from A to B. There are two routes that will bring you to B, one circuitous and the other direct. If you only have a single tank of fuel, opting for the direct route improves the likelihood you will arrive at your destination. An indirect approach to conservation is like taking the circuitous route, he says, and the chances are that you will run out of fuel. But if it's that simple, why are governments, non-governmental organisations, private bodies, and international organisations not jumping at the chance to experiment with this approach? Paying in Perpetuity There are those that have reservations about direct payments. The distinction between indirect and direct interventions is artificial, says Thomas Lovejoy, president of the Heinz Center, a nonprofit institution dedicated to improving the scientific and economic foundation for environmental policy. ‘In some cases, direct payment is the only way conservation can happen,’ he says. ‘In others, the indirect is important to reinforce a situation where there already is conservation. In yet others both are needed.’ Sjaak Swart of the Section of Science and Society at Groningen University in The Netherlands argues that if conservation is to succeed, it must be rooted in the hearts and minds of those involved. Direct payments create a vision of nature dominated by calculable, monetary concerns, he says. This approach can only work in the short term, he argues, and indirect tools like debate and education are needed to involve communities in the long term. ‘You need the commitment of the local people to save the biodiversity of our world,’ he says. Marine biologist Steve Trott agrees. He is project coordinator for the Local Ocean Trust, a charity-based conservation organisation operating in the Watamu and Malindi Marine Parks and Reserve in Kenya ( http://www.watamuturtles.com ), and is using direct payments to help reduce the slaughter of turtles by local fishermen. The Watamu Turtle Watch Program is currently paying fishermen just over $3 a turtle to release the animals from their nets rather than kill them. Before the scheme started in 2000, only around 50 turtles were being released from nets each year. By 2003, more than 500 a year were making it back into the sea. Elsewhere along the Kenyan coast, where fishermen do not get these payments, turtles continue to be killed, says Trott. However, the financial incentives are only part of a grander program of education and support to sensitise people to the conservation message, he says. Eventually, the plan is to stop payments altogether. ‘Payment will be reduced as education and awareness is increased to the point where it's phased out,’ he says. Reducing or stopping the payment could work, says Ferraro, but it is more likely that the turtles will begin to suffer once more. ‘If I had to wager, I'd bet people would go back to their old patterns eventually.’ This means that direct payments require an ongoing financial commitment, and many people don't like this idea, he says. To the Test The idea of direct payments needs empirical testing before it can be embraced with confidence, admits Ferraro. Funding bodies should demand experimental and control data to allow the success of an intervention to be gauged. Conservation biologists must therefore be trained in the skills needed to collect and evaluate these data. ‘Without adequate data and controls you're only going to be left with guesses and vague anecdotes about the effects of a program intervention,’ he says. Decision makers should begin to design controlled experiments from which they can make inferences about the effectiveness of these different interventions, he suggests. There are other drawbacks of direct payments. One concern is that they might just shift the pressure from one site to another that was not previously being exploited. Furthermore, in developing countries, land tenure is often ambiguous, which can make investment an unattractive prospect for funding agencies—they want to be sure they know where their money is going. But, notes Ferraro, such objections also apply to indirect interventions. ‘I don't necessarily believe that conservation payments will be successful,’ he says. ‘It's more I believe that of all the ideas out there for protecting biodiversity, this is the least bad.’ All this talk of cost, benefit, and efficiency is creeping into conservation speak. For some, these cold and calculating terms are an odd way to describe the world's wonderfully unpredictable wildlife. But, increasingly, there are calls for conservation biology to cast aside its sentimental demons: biodiversity is a commodity that can be bought and sold; conservation is business. Box 1. The Cucumber Conflict At the beginning of the 1990s, fishermen in the Galápagos began collecting sea cucumbers from the waters around their islands to meet ongoing demand for these aphrodisiac ‘earthworms of the sea’ in Southeast Asia ( Figure 3 ). Others intent on taking advantage of this commercial opportunity began to arrive from the Ecuadorian mainland in their hundreds. In 1998, Ecuador's president signed the Special Law of the Galápagos, which created the Galápagos Marine Reserve, protecting its waters from commercial fishing and imposed restrictions on domestic immigration. But by then, too many were already intent on reaping the financial rewards the sea cucumber promised them—by the end of the decade, a single sea cucumber could fetch nearly $2. Conservation biologists at the Charles Darwin Research Station on the central island of Santa Cruz worked out levels of fishing that might be sustainable. In 1999—the first season in which sea cucumber fisheries were monitored and regulated—nearly 800 fishermen collected more than 4 million animals worth more than $3.4 million in a short two-month window. In January 2000, fishermen protesting the closure of the fishery took over offices of the Galápagos National Park Service and Charles Darwin Research Station, holding humans and animals hostage. Figure 3 The Prized Galápagos Sea Cucumber, Stichopus fuscus Courtesy of Henry Nicholls. Box 2. Paying for Forests The longest-running and best-known example of a direct-payment initiative is the Programa de Pago de Servicios Ambientales (PSA) in Costa Rica ( Figure 4 ). In the second half of the 20th century, forest cover in Costa Rica fell from around 50% to 25%, and more than half of what remained was on unprotected, privately owned land. In 1996, the PSA was set up to make direct payments to individual landowners, associations of landowners, or indigenous reserves in exchange for ‘environmental services’—anything from forest conservation to providing a supply of water. Some 85% of contracts have been for forest conservation. By 2001, more than 2,800 km 2 were protected by payments of $4,000 per km 2 every year, and contracts covering a further 8,000 km 2 were being processed. Most of the money for these payments comes from a petrol tax on Costa Rican citizens, although the Global Environmental Facility has put up money for biodiversity conservation, Costa Rica's Office of Joint Implementation has paid for carbon sequestration, and domestic hydroelectricity and municipal water providers pay for water services. Figure 4 Forest Protected by Costa Rica's PSA Courtesy of Subhrendu Pattanayak. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516798.xml |
516773 | Spontaneous activity of rat pretectal nuclear complex neurons in vitro | Background Neurons in the mammalian pretectum are involved in the control of various visual and oculomotor tasks. Because functionally independent pretectal cell populations show a wide variation of response types to visual stimulation in vivo , they may also differ in their intrinsic properties when recorded in vitro . We therefore performed whole-cell patch clamp recordings from neurons in the caudal third of the pretectal nuclear complex in frontal brain slices obtained from 3 to 6 week old hooded rats and tried to classify pretectal neurons electrophysiologically. Results Pretectal neurons showed various response types to intracellular depolarizations, including bursting and regular firing behavior. One population of pretectal nuclear complex neurons could be particularly distinguished from others because they displayed spontaneous activity in vitro . These cells had more positive resting potentials and higher input resistances than cells that were not spontaneously active. The maintained firing of spontaneously active pretectal cells was characterized by only small variances in interspike intervals and thus showed a regular temporal patterning. The firing rate was directly correlated to the membrane potential. Removing excitatory inputs by blockade of AMPA and/or NMDA receptors did not change the spontaneous activity. Simultaneous blockade of excitatory and inhibitory synaptic input by a substitution of extracellular calcium with cobalt neither changed the firing rate nor its temporal patterning. Each action potential was preceeded by a depolarizing inward current which was insensitive to calcium removal but which disappeared in the presence of tetrodotoxin. Conclusions Our results indicate that a specific subpopulation of pretectal neurons is capable of generating maintained activity in the absence of any external synaptic input. This maintained activity depends on a sodium conductance and is independent from calcium currents. | Background Neurons in the mammalian pretectal nuclear complex (PNC) are involved in the control of various oculomotor reflexes, like the pupillary light reflex and the optokinetic reflex (OKR). Pupil constriction is controlled by neurons in the olivary pretectal nucleus that project bilaterally to the Edinger-Westphal nucleus [ 1 - 7 ]. Slow eye movements during OKR are generated by neurons in the nucleus of the optic tract (NOT) and in the adjacent dorsal terminal nucleus (DTN) of the accessory optic system (AOS) which project to the inferior olive (IO) and the nucleus prepositus hypoglossi [ 8 - 13 ]. In addition, PNC neurons carry signals related to saccadic eye movements to the dorsal lateral geniculate nucleus (LGNd) [ 14 - 16 ] and to the extrageniculate thalamus [ 17 ]. Other, reciprocal, projections connect the PNC to its contralateral counterpart, to the ipsilateral superior colliculus, and to the other AOS nuclei. The functions of those projections, however, are still under debate [ 18 - 23 ]. Each projection target receives input from an independent PNC neuronal population. Therefore, multiple retrograde tracing, e.g. from contralateral PNC and IO [ 24 ], or LGNd and Pulvinar [ 25 ], does not double label PNC cells. Furthermore, neurons with different projection targets show different response properties in vivo . Thus, neurons involved in the pupillary light reflex respond tonically to the overall retinal luminance [ 1 , 3 , 6 , 26 ]. OKR-related neurons are directionally selective in response to slow movements of large visual stimuli [ 8 , 27 - 33 ]. Finally, PNC neurons that project to thalamic visual centers only respond to fast moving visual stimuli without directional selectivity [ 15 , 17 , 25 , 34 - 36 ]. Furthermore, activity patterns of visual responses also differ significantly between PNC cell populations. Thus, saccade-related PNC neurons show short high frequency activity bursts, while luminance neurons or OKR-related neurons exhibit tonic activity at moderate firing levels. Although such differences to some extent directly reflect the response properties of specific input systems, different intracellular properties might enforce activity patterns provided by different input systems. We therefore studied intrinsic properties of rat PNC cells in vitro. In particular , we describe a population of cells in the caudo-lateral PNC that is characterized by intrinsically generated spontaneous activity in vitro , which is an unusual property for neurons in a sensory relay structure. Results In total, we obtained whole-cell recordings from 114 pretectal nuclear complex (PNC) neurons. Slices included the caudal part of the pretectum (Fig. 1 ), cells were recorded from the NOT, the posterior pretectal nucleus (PPN), and the olivary pretectal nucleus (OPN). Depolarizing current injections induced various spike patterns, like bursting (Fig. 2A,2B,2C ), non-adapting regular spiking (Fig. 2D ), or irregular spiking (Fig. 2E ). Usually, increasing the current amplitude also increased the firing rate, however, in about 31% (n = 35) of the cells, the firing rate showed a clear maximum in response to intermediate depolarizing current injections and decreased upon further depolarization (Fig. 2C ). Input resistances ranged from 201.0 to 776.3 MΩ (mean 410 ± 166.5 MΩ), resting potentials varied between -41.0 and -74.3 mV (mean -54.6 ± 8.5 mV). All cells tested, irrespective of the response type, responded to OT stimulation. Characteristics of spontaneous activity Of the cells recorded, 73 PNC neurons showed spontaneous firing at resting potential. Camera lucida reconstructions revealed that these cells were characterized by large fusiform cell bodies (diam. 15 μm and above) and multipolar dendritic trees that did not show any preference in their orientation. Whenever axons were also visible, they could be followed to leave the pretectal area in a ventro-lateral direction which indicates that these cells project to extrapretectal targets (arrowheads in Fig. 3A ). Their dendritic morphologies, however, did not allow to distinguish spontaneously active cells from PNC neurons that were not spontaneously active. Physiologically, all spontaneously active PNC neurons were characterized by a regular firing pattern when recorded at resting potential without any injected current (Fig. 3C ). The firing rate at resting potential of spontaneously active PNC cells varied between 0.9 and 9.4 imp/s (mean 3.0 ± 2.1 imp/s). Depolarizing current injections induced tonic firing patterns with only marginal adaptation (Fig. 3D ). Responses to hyperpolarizing current injections showed no sign of inward rectification. Furthermore, following cessation of hyperpolarizing current steps we never observed rebound spikes. Spontaneously active PNC cells on average had significantly higher input resistances (mean 454.1 ± 164.7 MΩ, p < 0.001), more positive resting potentials (mean -50.4 ± 7.0 mV, p < 0.001) and lower spike thresholds (mean -55.0 ± 3.96 mV, p < 0.001) than cells that did not show spontaneous activity (331.44 ± 137.1 MΩ, -58.4 ± 8.0 mV, and -40.66 ± 6.44 mV, respectively). In order to characterize the spike adaptation behavior of spontaneously active PNC cells, the holding potentials were increased in 5 mV steps by appropriate current injections in all recorded cells. In response to these depolarization steps, cells showed tonic increases of their firing rate without any sign of firing rate adaptation (Fig. 4 ). Also, no phasic firing rate increases were observed following the depolarizations. As could be already derived from current injections, the firing rate was directly correlated with the membrane potential. Increasing the membrane potential by positive current injections increased the firing rate until a maximum level was reached that could not be exceeded by further depolarization (Fig. 5A ). Consequently, when the firing rate is plotted against the membrane potential, the course of the resulting function is sigmoidal (Fig. 5B ). In order to get an impression about the regularity of the firing of spontaneously active PNC cells, interspike intervals (ISI) during maintained firing were analyzed in more detail (Fig. 6 ). Thus, maintained firing was recorded over a 10 s period at different membrane potentials and ISI histograms were generated from the recorded activity. ISIs obtained from these recordings followed a narrow unimodal Gaussian distribution with only little variation (Fig. 6B ). According to the correlation between the firing rate and the membrane potential, depolarization of the cells resulted in shifts of the maximum of the Gaussian distribution towards lower ISI values. Depolarization, however, did not change the shape of the distribution. The regularity of the maintained firing of spontaneously active PNC cells is also supported by autocorrelograms of the recorded spike trains (Fig. 6C ). The appearance of multiple equally spaced peaks in the autocorrelogram results from the regular timing of single spikes. Generation of spontaneous activity in vitro In order to test whether the spontaneous activity of PNC neurons in vitro depends on excitatory input, we first suppressed glutamatergic synaptic transmission and pharmacologically blocked AMPA receptors in 13 spontaneously active PNC cells (Fig. 7 ). As a control for the effectiveness of AMPA receptor blockade, the influence of the AMPA receptor antagonist CNQX on postsynaptic responses was monitored. In all cells tested, bath application of 20 μM CNQX resulted in a complete loss of EPSCs after electrical stimulation of optic tract afferent fibers (Fig. 7A,7E ). Although excitatory input was obviously blocked by CNQX application, the maintained firing remained unchanged (Fig. 7B,7F ). In particular, no drop in the firing rate was observed that could have been induced by a possible loss of excitatory input. Furthermore, the comparison of both the ISI distribution (Fig. 7C,7G ) and the autocorrelograms (Fig. 7D,7H ) obtained from spike trains before and during CNQX application did not show any significant difference. Hence, both the generation of spontaneous activity and its patterning seem to be independent from excitatory input via AMPA receptors. Similar results were achieved when NMDA receptors were blocked by bath application of 50 μM APV or when 2 mM kynurenic acid was applied to simultaneously block AMPA and NMDA receptors (N = 19). After having excluded glutamatergic synaptic inputs as a trigger for maintained firing, we tried to remove all synaptic input by adding cobalt to the extracellular medium in 12 spontaneously active PNC cells. This blocks the influx of calcium into the presynaptic terminal and thus prevents vesicular neurotransmitter release. Adding 1.5 mM CoCl 2 to the bath completely suppressed all electrically evoked postsynaptic currents (Fig. 8A,8D ) in all cells tested. In contrast to the complete loss of postsynaptic currents, however, the maintained firing always remained unchanged (Fig. 8B,8E ). As during glutamate receptor blockade, no reduction of the firing rate was observed that could have indicated the removal of an excitatory input. In addition, no increase of the firing rate appeared that could have indicated a loss in tonic inhibitory input regulating maintained activity. Finally, examination of the ISI distribution in the spike trains demonstrated that the patterning of the maintained activity also did not show any significant difference in the presence of Cobalt (Fig. 8C,8F ). This indicates that spontaneously active PNC cells generate their firing intrinsically without any external synaptic input. In current-clamp mode, each action potential was preceded by a depolarizing ramp (see, for example, Figs. 3C and 4 ). When cells were hyperpolarized to membrane potentials just below their resting potential single depolarizing ramps appeared that were not followed by an action potential. Concomitantly, in voltage-clamp mode, each unclamped action potential was preceded by an depolarizing inward current (Fig. 9A ). Because they did not disappear after substitution of calcium by cobalt in the external solution these current ramps were calcium independent. However, when 1 μM tetrodotoxin (TTX), a selective blocker of sodium channels, was added to the bath solution current ramps were eliminated together with the action potentials (Fig. 9B,9C ) in all seven cells tested. Discussion We have examined neurons in the rat PNC that are characterized by maintained activity in vitro . These spontaneously active PNC cells do not differ in their dendritic morphology from PNC cells that are not spontaneously active, but they show higher input resistances, more positive resting potentials, and lower spike thresholds. Furthermore, our results indicate that, firstly, all PNC cells that display spontaneous activity share firing characteristics, such as very regularly patterned spike trains and pure tonic firing rate increases in response to intracellular depolarizations. Secondly, the generation of the maintained firing of these cells is independent from excitatory synaptic input which suggests that these cells exhibit specific intrinsic properties that underly the generation of spontaneous activity. Finally, the patterning of the maintained firing is also independent from synaptic input, both excitatory and inhibitory, which indicates that their intrinsic membrane properties determine the firing pattern. To our knowledge, this is the first demonstration of spontaneous activity generated in vitro by cells in a subcortical visual relay structure. Generation of spontaneous activity in vitro Neurons that show spontaneous activity in vitro have been reported to exist in various mammalian CNS structures. Most extensively studied, spontaneously active neurons exist in the suprachiasmatic nucleus (SCN) which contains the biological clock that generates circadian rhythmicity. Thus, SCN cells not only generate spontaneous activity in vitro but they also maintain their circadian firing pattern [ 37 - 39 ]. Other populations of cells spontaneously active in vitro are the cholinergic interneurons in the neostriatum [ 40 ], dopaminergic cells in the substantia nigra [ 41 , 42 ], neurons in the subthalamic nucleus [ 43 , 44 ], neurons in the medial vestibular nucleus [ 45 - 47 ], cells from deep cerebellar nuclei [ 48 , 49 ], and cerebellar Purkinje cells [ 48 , 50 , 51 ]. Within the visual system, particularly the subcortical portion, spontaneous activity in vitro has been described to occur in thalamocortical neurons [ 52 ] and in isolated dopanimergic cells from the retina [ 53 ]. However, in the mammalian PNC cells that show maintained activity in vitro have not yet been reported. These cells are characterized by very regular firing pattern and monotonically increasing firing rates in response to intracellular depolarization. They differed from PNC neurons not spontaneously active by higher input resistance and more positive resting membrane potentials. Because glutamate receptor blockade did not change the firing characteristics, neither the firing rate nor the patterning of the firing, excitatory synaptic input through glutamate receptors seems unnecessary for the generation of the spontaneous activity. Furthermore, maintaining spontaneous firing in these PNC neurons also seems to be independent from synaptic input through other neurotransmitter systems because blockade of synaptic transmission by bath application of Cobalt did not change the firing. Thus, we conclude that the firing pattern is neither shaped by tonic excitation nor by tonic inhibition. Similar to spontaneously active cells in the structures noted above, our PNC neurons must possess intrinsic membrane properties that allow the generation of maintained activity. As far as the ionic mechanisms underlying the generation of spontaneous activity are concerned our results suggest that it critically depends on a TTX-sensitive sodium conductance. This sodium conductance leads to a steady inward current following spike afterhyperpolarization which induces membrane depolarization to spike threshold. This is similar to the ionic mechanism that is responsible for spike generation in spontaneously active neurons in the suprachiasmatic nucleus [ 54 ]. Because spontaneous firing in PNC neurons was unchanged by calcium substitution with cobalt the spontaneous activity generation seems independent from calcium conductances. Possible functional implications A characteristic response property of all spontaneously active PNC cells was that the firing rate increases to depolarizing voltage steps did not show any phasic components. This makes these cells perfectly suited to code maintained or tonic neuronal information. Of course, one has to keep in mind that neuronal response properties in vivo are shaped by numeous afferent input systems most of which are absent in the slice preparation. Thus, tonic inhibitory input could mask the maintained firing of spontaneously active PNC cells leading to a very different response pattern in vivo . Consequently, the maintained firing might become apparent only under very specific stimulus conditions upon withdrawal of the inhibitory input. However, from a functionl point of view, we regard it more reasonable to assume that PNC cells which are spontaneously active in vitro also exhibit tonic firing in vivo. Reviewing the known functions served by PNC neurons in vivo reveals only few reasonable suggestions for the possible functions which spontaneously active cells might accomplish. Thus, cells that typically show sustained activity in vivo are involved in the pupillary light reflex [ 2 , 3 , 6 , 26 , 55 ]. These cells are characterized by tonic increases of their firing rate to increases in the background luminance. However, these cells are predominantly found in the olivary pretectal nucleus (OPN) which is located in the rostro-medial PNC [reviewed in [ 56 ]]. Because our recordings were topographically restricted to the caudo-lateral PNC, particularly to the nucleus of the optic tract (NOT) and the posterior pretectal nucleus (PPN), it seems unlikely that we recorded from luminance neurons in the OPN. Neurons found in NOT and PPN include various functional cell populations. One of them has been associated with the generation of slow phase eye movements during OKR while others seem to transfer visual information linked to the execution of saccadic eye movements. Cells from these latter populations are all characterized by short duration, high frequency burst responses to fast image motions or rapid eye movements [ 15 , 17 , 25 , 34 - 36 , 57 ] and thus seem unlikely to correlate with cells that show maintained activity in vitro . Furthermore, because the timing of postsynaptic spikes with respect to their presynaptic input might be of considerable functional importance for saccade-related neurons such cells should exhibit lower input resistance than neurons for which spike time precision is less important. Low input resistances allow faster depolarization of the postsynaptic membrane and, hence, less temporal variance or "jitter" between presynaptic and postsynaptic spikes will occur. However, spontaneously active PNC cells on average showed higher input resistances in our sample and we therefore do not think that they represent saccade-related PNC neurons. On the other hand, cells that control compensatory eye movements during OKN are characterized by tonic firing in vivo when appropriately stimulated by low speed horizontal movements of whole field visual stimuli. In all mammals studied, neurons in the right PNC are excited by rightward stimulus motion and control eye movements to the right, while neurons in the left PNC are activated by leftward stimulus motion and control eye movements to the left [ 8 , 27 - 33 ]. The response properties of OKN-related PNC neurons to a large extent reflect response characteristics of their retinal afferents which are also activated by slow stimulus movements and show strong directional selectivity [ 58 ]. However, it may be functionally important to assure a constant level of maintained activity in OKN-related PNC neurons by additional intrinsic mechanisms in the absence of appropriate visual stimuli. Unilateral inactivation of PNC neuronal activity by focal injections of muscimol or lidocaine leads to spontaneous eye movements in darkness [ 11 , 59 ]. Because of the directional specificity in the PNC, inactivation of the right PNC elicits eye movements to the left, while inactivation of the left PNC elicits eye movements to the right. Thus, eye movements that appear after PNC inactivation seem to result from a distortion of a balanced activity between the two PNCs. Whenever the balance is distorted, premotor target structures postsynaptic to OKR-related PNC neurons receive stronger input from the PNC of one side and eye movements are elicited accordingly. It is therefore reasonable to assume that maintained activity spontaneously generated by OKR-related PNC assures this activity balance which is necessary for normal oculomotor function. In order to verify our hypothesis that OKR-related PNC neurons generate spontaneous activity that can be observed in vitro , it will be necessary to identify the postsynaptic targets of the spontaneously active PNC neurons. Conclusions We have been able to demonstrate a specific population of neurons in the PNC that is capable of generating spontaneous activity in vitro . The spontaneous firing depends on a sodium conductance and is independent from afferent synaptic input. Although the postsynaptic target and, consequently, the functional role of the spontaneously active PNC cells remain to be determined it is reasonable to assume that these cells also show spontaneous activity in vivo . Therefore, one likely candidate to represent spontaneously active cells in vivo are PNC neurons that are involved in the generation of slow compensatory eye movements during optokinetic nystagmus. If this is true, spontaneous firing might help to maintain an activity balance between neurons in the right and in the left PNC and thus stabilize eye position in the absence of retinal image motion. Methods Slice preparation Acute brain slices were obtained from 3 to 6 week-old Long-Evans hooded rats of either sex that had been raised at the institute's own colony. All experimental procedures were in strict compliance with governmental regulations and in accordance with the Guidelines for the Use of Animals in Neuroscience Research of the Society for Neuroscience. Animals were deeply anesthetized with halothane and a subcutaneous injection of ketamine (100 mg/kg body weight) and thiazine hydrochloride (1 mg/kg), and transcardially perfused with ice-cold artificial cerebro-spinal fluid (ACSF) containing (in mM), NaCl 123, KCl 2.5, NaH 2 PO 4 1, NaHCO 3 26, MgSO 4 1.3, CaCl 2 1.8, glucose 11, that was continuously gassed with 5% CO 2 / 95% O 2 . After the brain had been removed from the skull, 350 μ m-thick coronal slices were cut on a vibratome in ice cold ACSF. Three to four single slices that included the caudal PNC were obtained from each experimental animal. Slices were kept in ACSF at 36°C for at least one hour to allow recovery from the slicing procedure. For recording, they were transferred to a submerged type recording chamber where they were superfused at 3 ml/min with ACSF at 34°C during patch clamp experiments. Whole-cell patch clamp Whole-cell recordings from neurons in the caudo-lateral PNC were performed under visual guidance using infrared differential interference videomicroscopy [ 60 ]. For recording, borosilicate micropipettes (impedance 5–8 MΩ) were filled with internal solution composed of (in mM) potassium gluconate 130, sodium gluconate 5, HEPES 20, MgCl 2 4, Na 2 ATP 4, Na 3 GTP 0.4, EGTA 0.5, to which 0.5% biocytin was added for morphological single cell reconstruction. Measured membrane potentials were corrected for the junction potential of -10 mV. Postsynaptic responses were evoked with a concentric bipolar stimulation electrode (SNEX-100X, Rhodes Medical Instruments, Tujunga, CA) placed in the optic tract (OT) at the lateral PNC border. Electrical stimuli delivered were 0.5 to 2 mA in amplitude and had a duration of 100 to 500 μs. The neuronal signals were amplified and filtered using an EPC9 amplifier (Heka, Lambrecht, Germany), digitized at 20 kHz, and displayed, stored, and analyzed using PULSE/PULSEFIT software (Heka, Lambrecht, Germany). Unless otherwise stated, postsynaptic current responses evoked by OT stimuli were averaged over three consecutive stimulus applications. All drug effects are given as mean values ± standard deviation, they were statistically tested for significance using the Student's t-test. Drug delivery All drugs used were obtained from Sigma-Aldrich (Deisenhofen, Germany) and were bath applied. A10-minute application time proved sufficient to achieve stable responses. Application of 20 μM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) was used to block AMPA receptors. Either 50 μM APV or 2 mM kynurenic acid were used to block NMDA receptors. Na currents were suppressed by application of 1 μM tetrodotoxin (TTX). Histochemistry At the end of each recording session, slices were immersion fixed in 4% parafomaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C. After at least 24 h in fixative, slices were processed using standard histochemical techniques for visualization of biocytin with 3,3-diaminobenzidine (Sigma-Aldrich, Deisenhofen, Germany). Morphological reconstruction of stained cells was done with the aid of a camera lucida. Authors' contributions NP participated in the design of the study and executed all aspects including data collection, analysis and drafting the manuscript. MS initially designed the study, assisted with data collection and analysis, and edited the manuscript. Both authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516773.xml |
521685 | MAPK-dependent regulation of IL-1- and β-adrenoreceptor-induced inflammatory cytokine production from mast cells: Implications for the stress response | Background Catecholamines, such as epinephrine, are elaborated in stress responses, and mediate vasoconstriction to cause elevation in systemic vascular resistance and blood pressure. Our previous study has shown that IL-1 can induce mast cells to produce proinflammatory cytokines which are involved in atherogenesis. The aim of this study was to determine the effects of epinephrine on IL-1-induced proatherogenic cytokine production from mast cells. Results Two ml of HMC-1 (0.75 × 10 6 cells/ml) were cultured with epinephrine (1 × 10 -5 M) in the presence or absence of IL-1β (10 ng/ml) for 24 hrs. HMC-1 cultured alone produced none to trace amounts of IL-6, IL-8, and IL-13. IL-1β significantly induced production of these cytokines in HMC-1, while epinephrine alone did not. However, IL-6, IL-8, and IL-13 production induced by IL-1β were significantly enhanced by addition of epinephrine. The enhancing effect appears to involve NF-κB and p38 MAPK pathways. Flow cytometry showed the presence of β 1 and β 2 adrenoreceptors on resting mast cells. The enhancing effect of proatherogenic cytokine production by epinephrine was down regulated by the β 1 and β 2 adrenoceptor antagonist, propranolol, but not by the β 1 adrenoceptor antagonist, atenolol, suggesting the effect involved β 2 adrenoceptors. The enhancing effect of epinephrine on proatherogenic cytokine production was also down regulated by the immunosuppressive drug, dexamethasone. Conclusions These results not only confirm that an acute phase cytokine, IL-1β, regulates mast cell function, but also show that epinephrine up regulates the IL-1β induction of proatherogenic cytokines in mast cells. These data provide a novel role for epinephrine, a stress hormone, in inflammation and atherogenesis. | Background Atherogenesis involves the cellular infiltration of several cell types, including monocytes, T lymphocytes, and mast cells. Cytokine secretion by these cells and endothelial cells are contributing factors in the growth and propagation of atherosclerotic plaques as well as the stability and degradation of fibrous caps. Cytokines implicated in atherogenesis include Interleukin (IL)-1β, IL-6, IL-8, IL-13, and Tumor Necrosis Factor (TNF) [ 1 , 2 ]. IL-1β is secreted mainly by macrophages and virtually by every cell type in the body. IL-1β is produced in response to various stimulants, such as cytokines, bacteria, and viruses, but most interestingly to epinephrine [ 3 ]. IL-1β has a broad range of functions which includes activation of neutrophils, endothelial cells, monocytes, T-cells, and mast cells. It may also induce procoagulant changes in endothelial tissue. IL-6 induces an acute phase response consisting of increased fibrinogen synthesis and thrombocytosis with increased vascular permeability. The detection of IL-6 in the blood of patients suffering from unstable angina suggests that nuclear factor-kappa B (NF-κB) activation may be occurring at the vascular level in patients with heart disease [ 4 - 7 ]. IL-8 is in the CXC family of chemokines and functions to recruit neutrophils to the site of inflammation. IL-13 exerts multiple effects on cell differentiation and function of monocytes/macrophages. It can also suppress the cytotoxic function of monocytes/macrophages and the production of proinflammatory cytokines by these cells [ 8 , 9 ]. Mast cells are found preferentially around blood vessels and beneath the epithelium of the skin and mucus membranes [ 1 , 10 - 12 ]. Traditionally, mast cells are responsible for allergy and asthma pathogenesis. Typically, mast cell activation occurs in response to cross-linkage of the high affinity IgE receptor (FcεRI) by antigen and IgE [ 12 ]. Activation may also occur in response to a range of agents, such as pathogens, cytokines, and even oxidized low density lipoprotein (ox-LDL). After activation, key mediators secreted by mast cells include preformed mediators like histamine, proteoglycans, proteases, and several cytokines and growth factors [ 1 ]. Mast cells have been observed in both aortic atherosclerotic lesions and in coronary arteries. The large numbers of mast cells found in the adventitia of arteries and in the intima are in proportion to the severity of heart disease [ 13 ]. The study of the distribution, activation, and phenotype of mast cells in lesions of 250 specimens of human carotid arteries by Jeziorski, et al. further supports the role of mast cells in atherogenesis [ 14 ]. They demonstrated significant numbers and focal accumulations of mast cells in association with macrophages and extensive activation/degranulation at all developmental stages of atherosclerotic lesion development. It now appears likely that inflammatory events and mast cells play an important role in atherogenesis as recently reviewed by us [ 1 , 2 ]. Stress is known to influence immune function [ 15 - 17 ]. An immunoregulatory effect of the sympathetic nervous system in stress has been indicated for some time [ 18 ]. Catecholamines, such as epinephrine, norepinephrine, and dopamine, are elevated in stress responses, and mediate vasoconstriction and an increase in blood pressure as a result of increased peripheral vascular resistance. In disorders such as sepsis, cardiovascular disease, or cocaine abuse, catecholamines are elaborated in excess. Sustained increases in circulating catecholamines by infusion of epinephrine or norepinephrine have been shown to cause moderate cardiovascular and metabolic effects [ 19 ]. Catecholamines induce aggravation of aortic and coronary atherosclerosis in monkeys [ 20 ] and play a direct role in atherogenesis and cardiovascular disease [ 21 ]. Epinephrine and norepinephrine increase the uptake of low density lipoprotein in atheroscelotic plaques in rabbits and rats [ 22 ] as well as enhance proliferation of rat endothelial and smooth muscle cells [ 23 ]. It has been reported that norepinephrine increases adherence and chemotaxis of macrophages [ 24 ]. Epinephrine also upregulates the surface expression of L-selectin on monocytes in vitro [ 25 ]. Most recently, we have reported that nitric oxide production from macrophages induced by LPS is enhanced by catecholamines [ 26 ]. Both epinephrine and IL-1 are involved in acute phase responses seen in stress and in coronary artery disease. Studies have shown that norepinephrine can induce IL-1β mRNA in mycocardial tissue [ 27 ] and that infusion of IL-1β in animal models can induce expression of catecholamines [ 28 , 29 ]. These data suggest that, in some conditions, both IL-1β and catecholamines can be delivered to tissues that can then mediate additive or modulatory effects. Moreover, as reviewed by Gidron Y et al., [ 30 ] stress in conjunction with the release of catecholamines and proinflammatory cytokines, can potentiate atherogenesis. Hence, studies of the interactions between catecholamines, monokines and inflammatory cell activation are especially relevant. The aim of the study was to determine whether epinephrine affects IL-1β induced proatherogenic cytokine production in mast cells, a phenomenon previously not described. Our results indicated that epinephrine synergized with IL-1β in the production of proatherogenic cytokines, suggesting a potential role for this interaction in inflammatory and atherogenic states. Results Epinephrine enhances IL-1β-induced IL-6, IL-8, and IL-13 production in mast cells Human mast cell line, HMC-1, was incubated with IL-1β at various concentrations for 24 hours. The cell free supernatants of the cultures were harvested and subjected to IL-6 assay. HMC-1 cultured in medium alone produced trace amounts of IL-6. The IL-6 production from HMC-1 cultures treated with IL-1β was significantly increased in a dose-dependent manner (p < 0.0001) (Fig. 1 ). Since there was no significant difference in the IL-6 production induced by IL-1β at concentrations of 10 and 50 ng/ml, 10 ng/ml of IL-1β has been used to induce cytokine production in HMC-1 for the rest of the experiments. Epinephrine (Epi) alone at a concentration of 10 -3 M did not induce production of IL-6 in HMC-1 (Fig. 2 ). When epinephrine at 10 -3 to 10 -7 M concentration was added simultaneously with IL-1β into the cultures, the production of IL-6 was enhanced significantly (p < 0.05) compared with that induced by IL-1β alone (Fig. 2 ). Since the physiological concentration of epinephrine in plasma is 0.11 – 0.27 × 10 -6 M [ 31 ], we decided to use epinephrine at a supramaximal concentration of 1 × 10 -5 M for the rest of the experiments. In addition to IL-6, the enhancing effect of epinephrine was also observed in the production of IL-8 and IL-13 from IL-1β-induced HMC-1 cells (Fig. 3 ). Figure 1 IL-1β induces IL-6 production from HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 10 6 cells/ml) were cultured with IL-1β (1, 10, and 50 ng/ml) for 24 hours. The cultures were carried out in triplicate. Supernatants were harvested for measuring IL-6 by ELISA. By Student's t-test analysis, * indicates p < 0.0001, when compared with the medium alone. + indicates p < 0.0005, when compared with the IL-1 (1 ng/ml) group. Figure 2 Enhancing effect of epinephrine on IL-6 production from IL-1β-induced HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 10 6 cells/ml) were cultured with epinephrine (1 × 10 -3 to 1 × 10 -7 M) in the presence and absence of IL-1β (10 ng/ml) for 24 hrs in triplicate. Supernatants were harvested for measuring IL-6 by ELISA. By Student's t-test analysis, * indicates p < 0.05, when compared with the IL-1β-treated group. Figure 3 Effect of propranolol (Pro) and atenolol (Ate) on the enhancing effect of epinephrine (Epi) on production of IL-6 (A), IL-8 (B), and IL-13 (C) from IL-1β-induced HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 10 6 cells/ml) were cultured alone (Medium), or in the presence of IL-1β (10 ng/ml), Epi (1 × 10 -5 M), Pro (1 × 10 -4 to 1 × 10 -6 M), Ate (1 × 10 -4 to 1 × 10 -6 M), and the combinations of these reagents for 24 hrs in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA. IL-8 and IL-13 production were not detected in the Medium, Epi, Pro, and Ate alone groups. In A and B, by Student's t-test analysis, * and + indicate p < 0.05, when compared with the IL-1β-treated group, and the IL-1β plus Epi group, respectively. In C, * indicates p < 0.01, when compared with the IL-1β-treated group; p values for ++, +, ##, and # were <0.00005, <0.0005, <0.01, and <0.05, when compared with the IL-1β plus Epi group. To measure proatherogenic cytokine gene expression, HMC-1 were treated with IL-1β, epinephrine, and IL-1β plus epinephrine for 6 hours and harvested for transcriptional analysis via RT-PCR. IL-1β-treated HMC-1 showed increased IL-6 mRNA transcription as seen with densitometry, while epinephrine alone appeared to have no effect. When IL-1β and epinephrine were added together to HMC-1, IL-6 mRNA expression increased over IL-1β treatment alone (Fig. 4 ). The intensities of the cytokine and house keeping gene (HPRT) bands were measured by densitometry, and the ratio of the cytokine to the house keeping gene was calculated and assigned as the intensity index. The intensity indices for IL-6 were 0.36 for the control, 0.39 for IL-1β alone, 0.33 for epinephrine alone, and 0.54 for IL-1β plus epinephrine. IL-1β activated IL-8 mRNA production but epinephrine had no effect on IL-8 transcripts. IL-1β and epinephrine treatment together further increased IL-8 mRNA production (Fig. 4 ). The intensity indices for IL-8 were 0.17 for the control, 0.52 for IL-1β alone, 0.20 for epinephrine alone, and 0.64 for IL-1β plus epinephrine. The results with IL-13 expression showed the same pattern. IL-1β was a good inducer of IL-13 transcription while epinephrine alone only minimally induced IL-13 mRNA. The combined stimulus of IL-1β and epinephrine significantly increased IL-13 mRNA production over that seen with each stimulus alone (Fig 4 ). Intensity indices for IL-13 were 0.22 for the control, 0.57 for IL-1β alone, 0.20 for epinephrine alone, and 0.64 for IL-1β plus epinephrine. To evaluate further the ability of epinephrine to induce IL-13 transcription at a molecular level, we transiently transfected HMC-1 cells with minimal promoter sequences as described in the materials and methods. IL-1β at 10 ng/ml significantly increased IL-13 promotor activity as detected by luciferase expression (data not shown). Epinephrine did not enhance IL-13 promoter activity suggesting that post-transcriptional mechanisms may be involved in the IL-13 induction. It is likely that epinephrine either prolongs IL-13 mRNA half life and/or enhances IL-13 secretory processes from the mast cell in response to IL-1-stimulation. Figure 4 RT-PCR analysis for IL-6, IL-8, and IL-13 in HMC-1 treated with IL-1β and epinephrine. HMC-1 were treated for 6 hours with IL-1β with and without epinephrine and harvested for RNA preparation. RNA was subjected to RT-PCR with specific primers for target genes. HPRT was used as a house keeping gene to ensure equal loading. IL-6 gene expression was increased with IL-1β treatment and further increased with IL-1β plus epinephrine. Epinephrine alone had no effect on IL-6 gene expression in HMC-1. IL-8 and IL-13 showed similar results with a more robust expression of gene transcripts at this time point. Enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 is down regulated by adrenoceptor antagonists Since our previous study has shown that the effect of epinephrine on nitric oxide synthesis is mediated by β-adrenoceptors [ 26 ], β-adrenergic receptor antagonists (propranolol and atenolol) were used to block the enhancing effect of epinephrine on proatherogenic cytokine production in HMC-1. Propranolol (Pro) and atenolol (Ate) at a concentration of 1 × 10 -4 M did not affect the cell viability in the cultures (88 and 90%, respectively, while that of the medium control was 85%), nor induced production of IL-6, IL-8 or IL-13 (Fig. 3 ). When propranolol at 1 × 10 -4 and 1 × 10 -5 M was used in the culture, it significantly reduced the enhancing effect of epinephrine on IL-6 production (p < 0.05, Fig. 3A ). Propranolol at 1 × 10 -4 M also significantly reduced the enhancing effect of epinephrine on IL-8 production (p < 0.05, Fig. 3B ), and at 1 × 10 -4 and 1 × 10 -5 M significantly reduced the enhancing effect of epinephrine on IL-13 production (p < 0.00005 and 0.0005, respectively, Fig. 3C ). However, atenolol only significantly reduced the enhancing effect of epinephrine on IL-13 production (p < 0.05, Fig. 3C ), but not on IL-6 or IL-8 production (Fig. 3A and 3B ). Expression of β 1 and β 2 adrenergic receptors on mast cells In order to further identify whether the enhancing effect of epinephrine on proatherogenic cytokine production is through the β-adrenoceptor, HMC-1 cells were incubated with rabbit polyclonal antibodies against β 1 and β 2 adrenergic receptors followed by a FITC-labeled second antibody. By flow cytometry analysis, β 1 and β 2 adrenergic receptors were found in small amounts on HMC-1 (18.6 and 11.7% respectively) (Fig 5 ). Figure 5 Detection of β 1 and β 2 adrenergic receptors on HMC-1 cell by flow cytometry analysis. Resting HMC-1 were harvested and stained with a purified rabbit polyclonal antibody to either β 1 or β 2 adrenergic receptor and counter stained with a secondary goat anti-rabbit FITC conjugated antibody. Normal rabbit serum and the FITC conjugated goat anti-rabbit Ig G antibody was used as a staining control. Enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 is down regulated by immunosuppressants Since glucocorticoids are very effective treatment strategies for inflammatory disease, dexamethasone was used to determine its effect on atherogenic cytokine production in HMC-1. Dexamethasone (Dex, 1 × 10 -7 M) alone did not induce proatherogenic cytokine production (Fig. 6 ). However, Dex significantly inhibited the enhancing effect of epinephrine on IL-6 production (p < 0.05, Fig. 6A ). The cell viability of the cultures was not different between the medium control (70%) and Dex (64%) groups. Dex also significantly inhibited the enhancing effect of epinephrine on IL-8 and IL-13 production (p < 0.05, Fig. 6B and 6C ). When Dex was included in the IL-1β-treatment, it slightly decreased the cytokine production when compared to the IL-1β alone, but the decrease was not significant (Fig. 6 ). Figure 6 Effect of dexamethasone (Dex) on the enhancing effect of epinephrine (Epi) on production of IL-6 (A), IL-8 (B), and IL-13 (C) from IL-1β-induced HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 10 6 cells/ml) were cultured alone (Medium), or in the presence of IL-1β (10 ng/ml), Epi (1 × 10 -5 M), Dex (1 × 10 -7 M), and the combinations of these reagents for 24 hrs in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA. * p < 0.005, when compared with the medium control, + p < 0.05 compared to the IL-1β-treated group, and ++ p < 0.05 compared to the IL-1β plus Epi group. Role of NF-κB activation in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 NF-κB is an important transcription factor that mediates the transcription of many proinflammatory cytokine genes. To study the role NF-κB plays in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1, NF-κB activation was analyzed in HMC-1 cultures. NF-κB translocation, as seen by a shift in oligonucleotide binding in EMSA gels, was not seen in control or epinephrine treated cells (Fig. 7 ). A marked increase of NF-κB nuclear binding activity was observed in samples stimulated with IL-1β and IL-1β plus epinephrine for one hour but started to diminish after two hours (Fig. 7 ). Not only did IL-1β plus epinephrine have no further effects on NF-κB translocation over IL-1β treatment alone, it seemed to decrease after one and two hours of stimulation. Figure 7 Effects of IL-1β and epinephrine on NF-κB translocation in HMC-1. HMC-1 were treated for 1 and 2 hours with IL-1β and epinephrine. NF-κB translocation was analyzed by a shift in oligonucleotide binding in EMSA gels. After one hour of treatment, NF-κB translocation is increased in the IL-1β treated cells but not in the untreated or epinephrine treated cells. Addition of IL-1β plus epinephrine does not further enhance NF-κB translocation. After two hours of treatment, NF-κB translocation in HMC-1 starts to decrease. Role of p38 MAPK activation in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 Because of its importance in cytokine signaling, phosphorylated p38 MAPK was also assayed. After 30 minutes of activation, the HMC-1 were lysed to be analyzed for p38 activation by Western blot. The presence of phosphorylated p38 was greatly increased in the epinephrine and IL-1β plus epinephrine samples (Fig. 8 ). IL-1β alone had small effects on p38 activation at this time point while control levels were virtually nonexistent. Figure 8 Phosphorylated and total p38 MAPK in HMC-1 cells treated with IL-1β, epinephrine, and IL-1β plus epinephrine. HMC-1 were treated for 30 minutes with the indicated reagents and harvested for phosphorylated p38 expression by Western blot. Unphosphorylated p38 was used as loading control to show total MAPK expression. IL-1β treated cells showed a small amount of p38 activation while the bulk of p38 was activated with epinephrine. IL-1β plus epinephrine had no additional effects over epinephrine alone. Enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 is down regulated by NF-κB and p38 MAPK inhibitors To confirm the role of NF-κB and p38 MAPK in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1, Bay 11, an NF-κB inhibitor [ 32 ], and SB203580, a specific inhibitor of p38 MAPK [ 33 ], were added to the cultures. By themselves, neither Bay 11 (1 × 10 -5 M) nor SB203580 (1 × 10 -5 M) affected the cell viability of the cultures (92 and 87%, respectively, while that of the medium control was 92%), nor did they induce proatherogenic cytokine production (Fig. 9 ). However, Bay 11 and SB203580 significantly inhibited the enhancing effect of epinephrine on IL-6 production (p < 0.0005 and p < 0.00005, respectively, Fig. 9A ). Bay 11 decreased the IL-1β-epinephrine induced IL-8 production but not significantly, however SB203580 did significantly inhibit the enhancing effect of epinephrine on IL-8 production (p < 0.05, Fig. 9B ). Bay 11 and SB203580 significantly inhibited the enhancing effect of epinephrine on IL-13 production (p < 0.00005 and p < 0.0001, respectively, Fig. 9C ). Figure 9 Effect of Bay 11 and SB203580 on the enhancing effect of epinephrine (Epi) on production of IL-6 (A), IL-8 (B), and IL-13 (C) from IL-1β-induced HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 10 6 cells/ml) were cultured alone (Medium), or in the presence of IL-1β (10 ng/ml), Epi (1 × 10 -5 M), Bay 11 (1 × 10 -5 M), SB 203580 (1 × 10 -5 M), and the combinations of these reagents for 24 hrs in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA. IL-8 production was not detected in the Medium, Epi, Bay 11 alone groups, while IL-13 production was not detected in the Bay 11 and SB 203580 alone groups. In A, by Student's t-test analysis, * indicates p < 0.005, when compared with the IL-1β-treated group, and + and ++ indicate p < 0.0005 and <0.00005, when compared with the IL-1β plus Epi group. In B, * indicates p < 0.05, when compared with both the IL-1β-treated group, and the IL-1β plus Epi group. In C, * indicates p < 0.005, when compared with the IL-1β-treated group, and + and ++ indicate p < 0.00005 and <0.0001, when compared with the IL-1β plus Epi group. Discussion Inflammatory cytokines play an important role in atherogenesis. Acute phase response (APR) proteins have been demonstrated as risk factors for atherosclerotic heart disease [ 34 ]. Recent studies also suggest a prominent role for the APR in cerebrovascular disease and brain ischemia [ 35 ]. The APR culminates in the secretion of inflammatory cytokines such as IL-6, TNF-α", and IL-1 resulting in the synthesis of several proteins including C-reactive protein, fibrinogen, serum amyloid A protein, and ceruloplasmin [ 36 , 37 ]. These cytokines are intimately involved with the stress response [ 38 ]. These cytokines can also induce transcriptional regulation of complement genes that have been shown to play a role in cardiovascular disease [ 39 ]. Catecholamines are elaborated in stress responses which mediate vasoconstriction and elevate systemic vascular resistance and blood pressure. Catecholamines induce aggravation of aortic and coronary atherosclerosis in monkeys [ 20 ] and play a direct role in atherogenesis and cardiovascular disease [ 21 ]. Thus, it is important to understand the interaction between epinephrine and IL-1β with respect to atherogenic cytokine production. In this study, IL-1β, an acute phase cytokine, activated mast cells to produce proatherogenic cytokines, IL-6, IL-8, and IL-13, in a dose-dependent manner (Fig 1 , 2 , and 3 ). These results confirm our previous report that IL-1β regulates mast cell function [ 40 ]. These results also show that epinephrine significantly up regulated the IL-1β induction of proatherogenic cytokines in mast cells giving new insight into neuronal regulation of the immune system. The gene expression of these proatherogenic cytokines was also increased in IL-1β-induced HMC-1 cells by addition of epinephrine, suggesting that the enhancing effect of proatherogenic cytokine production is a result of increased cytokine gene transcription (Fig. 4 ). These data provide a novel role for epinephrine in inflammation and atherogenesis. IL-1β signaling probably synergizes with β 2 -adrenoreceptor-mediated signaling pathways in inducing proatherogenic cytokine production. Several reports have shown that the effect of catecholamines on immune function is due to β-adrenoceptors [ 41 - 45 ]. Flow cytometry data indicated that HMC-1 cells express both β 1 and β 2 adrenoceptors in small amounts (Fig. 5 ). The result showed that the enhancing effect of proatherogenic cytokine production by epinephrine is down regulated by β 1 and β 2 adrenoceptor antagonist, propranolol, but not by β 1 specific adrenoceptor antagonist, atenolol, further suggesting the enhancing effect involves β 2 adrenoceptors (Fig. 3 ). The down regulation by propranolol does not appear to be due to cytotoxicity of the antagonist since there is no difference in viabilities between the propranolol-treated and untreated cell cultures. It was interesting to see that propranolol caused a reduction of production of IL-13 to an amount that was much lower than that treated with IL-1β only (Fig. 3 ). It may be that epinephrine-induced enhancement of IL-13 production is more sensitive to the propranolol blocking. Activated NF-κB has been demonstrated in atheromatous plaques and has been shown to play a role in atherogenesis [ 46 ]. To study the mechanism of the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced mast cells, NF-κB and p38 MAPK activations were investigated. Control samples and epinephrine alone samples did not induce NF-κB activation. However, a marked increase in NF-κB activation was observed in samples stimulated with IL-1β and IL-1β plus epinephrine (Fig. 7 ). NF-κB activation was seen early at one hour and began to fade by two hours. NF-κB also was not increased by the addition of epinephrine to IL-1β and even seemed to decrease it at both time points suggesting that NF-κB is needed for cytokine induction but not for the enhancing effect. The presence of phosphorylated p38 MAPK was greatly increased in the epinephrine and IL-1β plus epinephrine samples but only minimally activated with IL-1β alone at a 30 minute incubation time point (Fig. 8 ). SB203580 blocked the IL-1β and IL-1β plus epinephrine effect on IL-6, IL-8, and IL-13 expression suggesting that p38 plays an important role in signaling from both IL-1β and epinephrine. The double stimulation of p38, early by IL-1β and later by epinephrine, may explain the enhancing effect on the production of IL-6, IL-8, and IL-13 in mast cells. The enhancing effect of epinephrine on proatherogenic cytokine production was also down regulated by immunosupressants, such as Dex. Dex at the concentration used in this study did not affect the cell viability of the culture, suggesting the down regulation effect of the drugs is not due to toxic effect. Dex also slightly, but not significantly, decreased IL-1β-induced cytokine production in mast cells (Fig. 6 ). Taken all together, these results indicate that β 2 -adrenoceptor antagonists and glucocorticoids may have clinical potential in stress-mediated disease and atherogenesis. All the signaling pathways induced by IL-1β and epinephrine in mast cells are complex and beyond the scope of this manuscript. However, two important inflammatory pathways, NF-κB and p38 MAPK, have been shown. IL-1β release from immune challenge and epinephrine elevated from stress response can jointly stimulate mast cells to increase IL-6, -8, and -13 production above that which is seen with either stimulus alone. The exact mechanisms are unclear, but we have shown that IL-1β is a strong inducer of NF-κB while epinephrine is a strong inducer of p38 MAPK. Neither NF-κB nor p38 MAPK was activated further by IL-1β plus epinephrine compared to either stimulus alone nor was the promotor activity of IL-13 increased by the double stimulus as seen by luciferase activity of a IL-13 reporter gene construct. These data would suggest that IL-1β is activating IL-6, IL-8, and IL-13 by NF-κB while p38 MAPK activation is enhancing protein production by inducing other transcription factors, stabilizing the gene mRNA, or other forms of post-translational modification. These mechanisms are summarized in Fig. 10 . Figure 10 Schematic presentation showing the possible route of IL-6, IL-8, and IL-13 signaling. Endogenous IL-1β production may occur with immune challenge by cytokines, bacteria, and viruses, and any microtrauma in the body while epinephrine is released in states of stress or sympathetic nervous system activation. The pathways activated by these signals converge on IL-6, IL-8, and IL-13 genes to induce cytokine production that is greater than either signal alone. IL-1β activates the NF-κB pathway which leads to significant amounts of IL-6, IL-8, and IL-13 production. Epinephrine activates the p38 MAPK pathway which may activate other transcription factors or stabilize the IL-6, 8, and 13 mRNA. From our data it is evident that IL-1β and epinephrine do not combine to further activate NF-κB or the promotor activity of the IL-13 gene. The importance of IL-6, IL-8, and IL-13 are listed in the figure. Conclusions In conclusion, stress related catecholamines, such as epinephrine, synergized with IL-1β in gene expression and production of proatherogenic cytokines, IL-6, IL-8, and IL-13 in mast cells. The enhancing effect of proatherogenic cytokine production by epinephrine on IL-1β-induced mast cells was down regulated by β-adrenoceptor antagonist, propranolol, and the immunosuppressant Dex. These data support a novel role for catecholamines in disorders such as inflammation and atherogenesis. These data also indicate that β-adrenoceptor antagonists and immunosuppressants may be used preventively and therapeutically for modulation of the catecholamine – proatherogenic cytokine axis in disease states. Methods Mast cell culture and the induction of cytokine production in HMC-1 cells HMC-1 cell line, established from a patient with mast cell leukemia, were graciously provided by Dr. Butterfield (Mayo Clinic, Rochester, MN). These cells were maintained in RPMI 1640 media (GibcoBRL, Rockville, MD), supplemented with 5 × 10 -5 M 2-mercaptoethanol (Sigma Chemical Company, St. Louis, MO), 10 mM HEPES (GibcoBRL), Gentamycin 50 μg/ml, 5 μg/ml insulin transferrin, 2 mM L-glutamine, and 5% heat inactivated fetal bovine serum (Atlanta Biologicals, Atlanta, GA), at 37°C and in 5% CO 2 mixture [ 33 ]. HMC-1 cells were cultured and maintained in 25 cm 2 flasks. To each well of a 6 well culture plate, two ml of HMC-1 mast cells at 0.75 × 10 6 cells/ml concentration were cultured with epinephrine at 1 × 10 -5 M concentration in the presence and absence of IL-1β (10 ng/ml) for 24 hrs. The cultures were carried out in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA and cell viability and numbers of the culture were analyzed. ELISA for cytokine proteins Cytokine ELISA was performed for the following cytokines: IL-6, IL-8, and IL-13. ELISA was carried out on cell-free culture supernatants using commercially available ELISA kits, according to manufacturers instructions as earlier described (R&D Systems, Minneapolis, MN; Immunotech, Westbrook, ME; Genzyme, Cambridge, MA). Results were analyzed on an ELISA plate reader (Dynatech MR 5000 with supporting software) [ 47 , 48 ]. Measurement of cell viability of the cultures At the end of incubation, the cells were subjected to the viability count by trypan blue (TB) dye exclusion technique. Two tenths ml of cell cultures were mixed with 0.05 ml of TB, applied to hemocytometer, and counted under a microscope. The cell viability is calculated by dividing the number of live cells (unstained cells) by the total number of all cells (TB-stained and unstained cells) and expressed as a percent. Analysis of cytokine gene expression by RT-PCR HMC-1 were treated with the appropriate reagents and allowed to incubate at 37°C before being harvested for RNA. RNA was extracted from HMC-1 (3 × 10 6 cells) by the addition of 1 ml of RNAzol B (Tel-Test, Inc., Friendswood, Texas) [ 49 ]. After shaking for 1 minute the samples were centrifuged at 12,000 × g for 15 minutes at 4°C. The aqueous layer was washed twice with 0.8 ml phenol : chloroform (1:1, v/v), centrifuged at 12,000 × g for 15 minutes at 4°C, washed once with 0.8 ml of chloroform and centrifuged at 12,000 × g for 15 minutes at 4°C again. Isopropanol was added to the aqueous phase, and the preparation was frozen at -20°C overnight. The following day, the samples were centrifuged at 12,000 × g for 30 minutes at 4°C. The RNA pellet was washed with 1 ml 75% ethanol and allowed to air dry until all moisture was gone. The pellet was resuspended in DEPC water and quantitated by optical density readings at 260 nm. cDNA was synthesized with murine leukemia virus reverse transcriptase (2.5 U/μl), 10 × PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3), 1 mM each of the nucleotides dATP, dCTP, dGTP and dTTP; RNase inhibitor (1 U/μl), MgCl 2 (5 mM), and oligo(dT) 16 (2.5 μM) as a primer. The samples were incubated at 42°C for 20 minutes, 99°C for 20 minutes, and 5°C for 5 minutes in a DNA thermocycler (Perkin-Elmer Corp., Norwalk, CT) for reverse transcription. PCR of cDNA was done with MgCl 2 (1.8 mM), each of the dNTPs (0.2 mM), AmpliTaq polymerase (1 U/50 μl), and paired cytokine-specific primers (0.2 nM of each primer) to a total volume of 50 μl. Cycles consisted of 1 cycle of 95°C for 2 min, 35 cycles of 95°C for 45 sec, 60°C for 45 sec, and 72°C for 1 min 30 sec, and lastly, 1 cycle of 72°C for 10 min. Ten microliters of the sample were electrophoresed on a 2% agarose gel and stained with ethidium bromide for viewing. Primer sequences used are as follows: HPRT: 5' CGA GAT GTG ATG AAG GAG ATG G 3' and 5' GGA TTA TAC TGC CTG ACC AAG G 3'; IL-6: 5' ATG AAC TCC TTC TCC ACA AGC GC 3' and 5' GAA GAG CCC TCA GGC TGG ACT G 3'; IL-8: 5' ATG ACT TCC AAG CTG GCC GTG GCT 3' and 5' TCT CAG CCC TCT TCA AAA ACT TCT C 3'; and IL-13: 5' GGA AGC TTC TCC TCA ATC CTC TCC TGT T 3' and 5' GCG GAT TCG TTG AAC CGT CCC TCG CGA AA 3'. Densitometry was done by normalizing target genes to house keepers using Un-Scan-It Version 5.1 software (Orem, UT). The PCR experiment was repeated twice. NF-κB assay in HMC-1 HMC-1 were stimulated with PMA, IL-1β and/or epinephrine and then harvested for EMSA analysis [ 49 , 50 ]. Cells were washed with PBS and mixed with one hundred microliters of hypotonic buffer which contains: 10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM aprotinin, 1 μM pepstatin, 14 μM leupeptin, 50 mM NaF, 30 mM β-glycerophosphate, 1 mM Na 3 VO 4 , and 20 mM p-nitrophenyl phosphate. Cells were incubated over ice for 30 minutes and then vortexed after the addition of 6.25 μl of 10% of Nonidet P-40. After 2 minutes of centrifugation at 30,000 × g, supernatants were kept at -80°C while the pellets were collected and vortexed every 20 minutes for 3 hours in 60 ml of a hypertonic salt solution: 20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 12 mM DTT, 1 mM PMSF, 1 μM aprotinin, 1 μM pepstatin, 14 μM leupeptin, 50 mM NaF, 30 mM β-glycerophosphate, 1 mM Na 3 VO 4 , and 20 mM p-nitrophenyl phosphate. Nuclear translocation of NF-κB was analyzed by the Electrophoretic Mobility Shift Assay (EMSA) using the nuclear fraction. Seven micrograms of nuclear protein were added to 2 ml of binding buffer (Promega, Madison, WI), and 35 fmol of double stranded NF-κB consensus oligonucleotide (5' AGT TGA GGG GAC TTT CCC AGG C 3') (Promega, Madison, WI) end labeled with γ-P 32 ATP (Amersham Biosciences, Piscataway, NJ). The samples were incubated at room temperature for 20 minutes and run on a 5% nondenaturing polyacrylamide gel for 2 hours. The gel was then dried on a Gel-Drier (Bio-Rad Laboratories, Hercules CA) and exposed to Kodak X-ray film at -80°C. Detection of p38 MAPK by Western blot Cells were treated and lysed in lysis buffer (50 mM Tris HCL, 150 mM NaCl, 1 mM EDTA, 1% Triton × 100, and 0.1% SDS) to be analyzed for p38 MAPK activation by Western blot [ 29 ]. Briefly, 50 μg of sample protein was diluted 1:2 with Laemmli buffer (Bio-Rad laboratories, Hercules, CA) and boiled for 10 minutes in a sand bath. The resultant sample was then run in a Bio-Rad Modular Mini Electrophoresis System (Hercules, CA) on a 10% polyacrylamide gel for 1 hour and then transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad laboratories, Hercules, CA) for 1 hour. The blot was then incubated in blocking buffer (1% BSA and 0.1% Tween in PBS) for 1 hour at room temperature with gentle agitation. Rabbit anti-human Phospho-p38 MAPK (Thr180/Tyr182) polyclonal antibody (Calbiochem, San Diego, CA) was diluted 1:1000 in blocking buffer and incubated on the blot overnight at 4°C with gentle agitation. After the primary antibody was removed the blot was washed three times for 10 minutes each with agitation in the wash buffer (0.1% Tween in PBS). The blot was then incubated in horse radish peroxidase conjugated mouse anti-rabbit Ig's antibody (human adsorbed, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:5000 in blocking buffer. The blot remained in the secondary antibody for 1 hour at room temperature. The blot was then washed again and covered with Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) for 5 minutes. The blot was then exposed to acetate transparency film (Kodak, Rochester, NY) and developed. The same protocol was repeated for total p38 MAPK analysis. Analysis of β-adrenoceptor by flow cytometry Resting HMC-1 were centrifuged, washed in PBS at room temperature, and resuspended in 100 μl of PBS. The cells were incubated for 20 minutes with rabbit polyclonal anti β 1 or β 2 adrenergic receptor antibodies (Santa Cruz, Santa Cruz, CA) using normal rabbit serum as a control. The samples were washed with PBS with 0.01% sodium azide and resuspended in 100 ml PBS. FITC labeled goat anti-rabbit Ig's antibody was added to the samples and allowed to bind for 20 minutes. The samples were once again washed with PBS with 0.01% sodium azide and resuspended in 100 μl of PBS. In addition, HMC-1 were pretreated with normal rabbit serum and incubated with FITC labeled goat anti-rabbit Ig's antibody as a control for nonspecific binding [ 51 ]. Cell suspensions were then gated and analyzed based on fluorescence using a Becton Dickinson FACSCalibur 4-color flow cytometer (San Diego, CA) and histograms generated on WinMDI 2.8 software (kindly provided by Joseph Trotter over the internet). IL-13 promotor analysis HMC-1 were treated with IL-1β (10 ng/ml), epinephrine (10 -5 M), and IL-1β plus epinephrine to investigate IL-13 promotor activity. Untreated cells were used as a control. Transient transfection assays were performed using a reporter gene construct containing the minimal promoter sequence of IL-13. The promoter sequence (-233 to + 50, relative to the transcription initiation site) of the IL-13 gene was fused to the luciferase coding sequence. Plasmid DNA was obtained with double-cesium chloride purification (BioServe Biotechnologies, Laurel, MD), while SuperFect reagent (Qiagen) was used for transient transfections of HMC-1 cells. Two micrograms of plasmid DNA and 8 μl SuperFect reagent were used for transfection of 1 × 10 6 HMC-1 cells. Luciferase expression was monitored by chemiluminescence of cell lysates 24 hrs after transfections using the Enhanced Luciferase Assay Kit (Analytical Luminescence Laboratory, Ann Arbor, MI). Statistical analysis of the data All experiments were done in triplicate. The data were analyzed by Student's two-tailed t -test using Statistica software (StatSoft, Inc., Tulsa, OK). All data were reported as means ± SE. A p -value of less than 0.05 was considered significant. List of abbreviations used HMC-1, human mast cell – 1 Epi, epinephrine Pro, propranolol Ate, atenolol Dex, dexamethasone MAPK, mitogen-activated protein kinase Author's contributions DSC designed experiments, oversaw research, and wrote paper. SMF designed and conducted experiments and wrote paper. SP helped with experiments. KC conducted experiments. EK conducted experiments. SAL conducted experiments. SKH conducted experiments. GK oversaw research. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521685.xml |
544948 | JNER: a forum to discuss how neuroscience and biomedical engineering are reshaping physical medicine & rehabilitation | Advances in neuroscience and biomedical engineering deeply affect the clinical practice of physical medicine & rehabilitation. New research findings and engineering tools are continuously made available that have the potential of dramatically enhancing the ability of clinicians to design effective rehabilitation interventions. This quickly evolving research field is difficult to track because related literature appears in a wide range of scientific journals. There is a need for a scientific journal that offers to its readership a forum at the intersection of neuroscience, biomedical engineering, and physical medicine & rehabilitation. The Journal of NeuroEngineering and Rehabilitation ( JNER) is intended to fill this gap and foster cross-fertilizations among these disciplines. By making readily available to clinicians selected studies with potential impact on physical medicine & rehabilitation, JNER is anticipated to foster the development of novel and more effective rehabilitation strategies. Conversely, by presenting clinical problems to a readership of neuroscientists and engineers, JNER is expected to generate innovative work in neuroscience and biomedical engineering with future applications to physical medicine & rehabilitation. JNER will leverage on Open Access as a means to guarantee that its content is readily available to scientists, clinicians, and the general public thus promoting scientific and technological advances that are relevant to rehabilitation. JNER is an Open Access initiative. Open Access assures dissemination to the widest possible audience and is seen by many as essential for publicly funded research. BioMed Central offers an outstanding platform to make JNER possible and allow neuroscientists, biomedical engineers, and clinicians to see their work published in a timely manner and thus make an immediate impact in the field of rehabilitation. JNER will focus on innovative work with higher likelihood of a dramatic impact on rehabilitation. Thus, priority will be given to outstanding and visionary scientific reports, i.e. those proposing exceptionally innovative concepts with great potential in the field. | A new journal for a quickly evolving research field During the past decade, we have witnessed profound changes in physical medicine & rehabilitation originated by advances in neuroscience and biomedical engineering. For example, imaging and neurological assessment methods have dramatically improved the management of patients with motor impairments; robotics and artificial muscle research have generated revolutionary concepts in orthotics and prosthetics; and advances in cortical recordings and the understanding of central nervous system mechanisms have changed the way clinicians look at movement disorders. These techniques and others have brought about, and will continue to give rise in the future to, dramatic advances in physical medicine & rehabilitation. As advances in neuroscience and biomedical engineering continue to generate new techniques, with tremendous impact in the field of physical medicine & rehabilitation, it becomes apparent that there is an urgent need for establishing an outlet for the intersection of these three research fields. Journal of NeuroEngineering and Rehabilitation (JNER) aims to provide such an outlet, hosting the introduction of new methods and the discussion of their clinical implications, and offering an opportunity to publish, in a timely manner, articles relevant to the cross-fertilization of neuroscience, biomedical engineering, and physical medicine & rehabilitation. JNER's editorial board [ 1 ] demonstrates the commitment of the journal to interdisciplinary research and international representation. Members of the editorial board are leading scientists working in different parts of the world in the research areas of neuroscience, biomedical engineering, and physical medicine & rehabilitation. They share an interest in scientific work that has potential impact on clinical practice in physical medicine & rehabilitation and an enthusiasm for Open Access. The editorial board is pleased to become a part of the growing group of institutions and individuals who work to promote Open Access – BioMed Central currently publishes over 100 Open Access journals covering all areas of biology and medicine, and has over 450 institutional members from about 40 countries. Open access to advance science and clinical practice JNER's Open Access policy changes the way in which articles are made available to the scientific community. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [ 2 ]. Third, a copy of the full text of each Open Access article is permanently archived in online repositories separate from the journal. JNER's articles are archived in PubMed Central [ 3 ], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [ 4 ] in Germany, at INIST [ 5 ] in France and in e-Depot [ 6 ], the National Library of the Netherlands' digital archive of all electronic publications. Open Access has four broad benefits for science and the general public. First, authors are assured that their work is disseminated to the widest possible audience, given that there are no barriers to access their work. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. It has been suggested that free online articles are more highly cited because of their easier availability [ 7 ]. Second, the information available to researchers will not be limited by their library's budget, and the widespread availability of articles will enhance literature searching [ 8 ]. Third, the results of publicly funded research will be accessible to all taxpayers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Note that this public accessibility may become a legal requirement in the US if the proposed Public Access to Science Act is made law [ 9 ]. Fourth, a country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones (although creating access to the internet is another matter [ 10 ]). Open Access will increasingly become an accepted way to disseminate information to the scientific community and the public at large. By becoming part of the movement for Open Access, JNER will contribute to make the latest advances in neuroscience and biomedical engineering, which have the potential to impact on the clinical practice of physical medicine & rehabilitation, readily available to scientists, clinicians, and the general public. Because of its inherent interdisciplinary nature, JNER will foster further advances in the field thanks to the cross-fertilization among science, technology, and clinical practice. Science and technology are expected to offer new tools to design clinical interventions and, vice versa, clinical problems are anticipated to foster basic research in neuroscience and the development of new technologies. Besides, increased awareness of the way science and technology can improve clinical outcomes will lead to better quality of healthcare in rehabilitation. Changes currently occurring in this field are so dramatic that we expect, in a few years, that modernized rehabilitation inpatient and outpatient units will be completely different from what is the state-of-the-art today. For instance, we envision that continuous monitoring of patient status will be performed via miniature, wireless, wearable sensors which not only allow clinicians to monitor vital signs, but also track motor activities and provide a means to analyze motor patterns associated with recovery. Furthermore, robotic devices will be used to enhance physical therapy, ad hoc protocols will be designed for each patient, and augmented and virtual reality tools will enhance rehabilitation by becoming part of routine exercise protocols. Outstanding and visionary articles make the difference Open Access to outstanding and visionary scientific reports appears to be a tremendous tool to increase the speed at which clinical practice changes as a result of advances in neuroscience and biomedical engineering. By prioritizing outstanding and visionary publications, JNER intends to provide a forum for ideas and concepts that could make a difference in physical medicine & rehabilitation by innovating the design of clinical interventions. Publication in JNER is free for the first 6 months following the launch of the journal. Manuscripts submitted after this period will be subject to an article-processing charge on acceptance. Waiver requests will be considered on a case-by-case basis, by the Editor-in-Chief. Authors can circumvent the charge by getting their institution to become a 'member' of BioMed Central, whereby the annual membership fee covers the article processing charges for authors publishing in any of the BioMed Central journals. Current members include NHS England, the World Health Organization, the US National Institutes of Health, Harvard, Princeton and Yale universities, and all UK universities [ 11 ]. No charge is made for articles that are rejected after peer review. Many funding agencies have also realized the importance of Open Access publishing and have specified that their grants may be used directly to pay article-processing charges [ 12 ]. The article-processing charges pay for efficient and thorough peer review, for the article to be freely and universally accessible in various formats online, and for the processes required for inclusion in PubMed and archiving in PubMed Central, e-Depot, Potsdam and INIST. Funding available to JNER's editorial board will be solely used to further promote the journal and to continuously increase the scientific quality of JNER's articles. The first articles published in the journal demonstrate the commitment of JNER to high quality, prioritizing visionary work, and focusing on research that has the potential of a great impact on physical medicine & rehabilitation. Forthcoming articles will further prove such commitment. Topics of interest to come in the next few months are virtual and augmented reality in rehabilitation, wearable technology in rehabilitation, methods for the analysis of movement, and robotics applied to rehabilitation. These are all topics of great relevance for the research at the intersection of neuroscience, biomedical engineering, and physical medicine & rehabilitation. Special thanks to the authors of the first articles published in JNER as well as to the authors who submit their manuscripts in the future and support our journal and the Open Access initiative. Members of the editorial board and reviewers have done some excellent work; special thanks to them and the Managing Editor, Sara Midwood, for their contributions to JNER . | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544948.xml |
393293 | Annotation Marathon Validates 21,037 Human Genes | null | The announcement of the human genome sequence three years ago was widely hailed as one of the great scientific achievements in modern history, and with good reason. Determining the structure and nature of the genetic code promises to provide valuable insights into human evolution and the molecular basis of disease. But sequencing the genome is just the first step toward this decidedly worthy goal—the monumental task of ascribing biological meaning to those sequences has just begun. And while researchers know a great deal about some of the 30,000 or so genes in the human genome, they have yet to ascribe function to the majority of them. Takashi Gojobori and a large international team of collaborators have now taken a big step toward narrowing this knowledge gap. Scientists at the annotation “marathon” of 41,118 cDNA clones Deciphering the human genome presents such a daunting challenge in part because it's so huge, making it difficult to distinguish genetic signal from noise. Simpler organisms have much more compact genomes. In the case of brewer's yeast, for example, genes that encode proteins account for about 70% of the genome. In contrast, only about 1% to 2% of the human genome codes for proteins. That translates to about one gene for every 2,000 bases for yeast compared to about one gene for every 150,000 bases for humans. The low density of human genes makes identifying them difficult enough, but this process is further complicated by how genes are organized in the human genome. The functional parts are broken up into smaller segments called exons, which are separated in the genome by intervening sequences called introns. This configuration also occurs in simpler organisms, but since the number and size of introns is relatively small in simpler organisms, it's easier to tell what's a gene and what isn't. In humans, the introns are extremely long, as are the gaps between the genes, and the exons are tiny in comparison; plus, it takes many more of these short, scattered exons to make one gene. One approach to this problem is to use computer algorithms that scan the genome sequence looking for segments of DNA sequences that could potentially encode proteins. Gojobori and colleagues, however, used a different approach. They analyzed the sequences of 41,118 full-length cDNAs available from six sequencing centers around the world. These cDNAs are stretches of DNA that represent genes that have already been expressed and used by the cell for protein production. Since all the exons have been spliced together and the introns removed, these cDNAs correspond to the functional versions of these genes, allowing researchers to work backward, looking for the sequences in the genome. In order to process the 41,118 cDNAs, the researchers used a combination of computer algorithms and expert human analysis. To tackle such an enormous project, 158 genome scientists, representing 67 institutions from 12 countries, gathered in Japan in the summer of 2002. Over the course of a ten-day annotation marathon, the scientists validated, mapped, and annotated the cDNAs. As things stand, the team has been able to assemble the cDNAs into over 20,000 strong candidates for human genes. From just the initial analysis of the data generated by this group, several valuable findings about the human genome have emerged: there are over 5,000 candidates for new genes, including an exciting group of several hundred that do not appear to encode proteins; up to 4% of the genome appears not to be represented in the current human genome sequence; and several thousand DNA sequence variants have been uncovered that will be useful for disease mapping studies. But perhaps most important of all, the data from this study have been collected and assembled into a large searchable database called H-Invitational Database, which is linked to other functional databases around the world. This will be an invaluable resource for geneticists, and will serve as a starting point for further analyses. Future research on the human genome will be aimed at expanding the list of known genes and analyzing the properties of these genes. This study not only moves us closer to a complete functional description of the human genome, it also builds on the traditions of international cooperation and large-scale collaboration that played such an important part in deciphering the sequence itself. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC393293.xml |
546187 | Impact of hiatal hernia on histological pattern of non-erosive reflux disease | Background Hiatus hernia (HH) has major pathophysiological effects favoring gastroesophageal reflux and hence contributing to esophageal mucosa injury, especially in patients with severe gastroesophageal disease. However, prospective studies investigating the impact of HH on the esophageal mucosa in non-erosive reflux disease (NERD) are lacking. This study evaluated the association between the presence of (HH) and the histological findings in symptomatic patients with NERD. Methods Fifty consecutive patients with gastroesophageal reflux disease (GERD) were enrolled. After conventional endoscopy, Lugol solution was applied and biopsy specimens were obtained. Histological parameters including basal zone hyperplasia, papillary length and cellular infiltration were evaluated. The chi-square test with Yates' correlation was used for comparing discrete parameters between groups. However, Fisher's exact probability test was used where the expected frequencies were lower than 5. Wilcoxon's test for unpaired samples was preferred in cases of semi-quantitative parameters. Results The presence of HH along with more severe findings (0.01 < P < 0.05) was confirmed in 18 patients. NERD was observed in 29 (58%) patients. Basal zone hyperplasia and loss of glycogen accompanied HH in all cases, and the correlation was significant in NERD ( P < 0.001). The remaining histological patterns were similar between erosive reflux disease and NERD in the presence of HH. Conclusion The presence of HH is correlated with more severe endoscopy findings, and predisposes for severe histological abnormality in cases of NERD. | Background Gastroesophageal reflux disease (GERD) is a common condition that affects 25–30% of the population [ 1 ]. It clearly involves multifactorial pathophysiology, yet the factors underlying why only some patients develop reflux esophagitis are unclear [ 2 ]. Symptoms and demographic data do not allow differentiation between the endoscopy-negative (non-erosive reflux disease; NERD) and endoscopy-positive (erosive reflux disease; ERD) forms of the disease. In fact most patients with typical symptoms of GERD have normal esophageal mucosa on upper endoscopy. Indeed, more than two-thirds of all patients with reflux symptoms never develop esophageal erosions, ulcers or strictures [ 3 ]. This group of NERD patients constitutes a significant clinical problem since they appear to be relatively resistant to proton-pump inhibitors (PPIs) [ 4 , 5 ]. Hiatal hernia (HH) has been considered to be one of the pathophysiological mechanisms that contributes to the development of GERD, promoting refluxate access and impaired acid clearance; however, the impact of this mechanism in NERD is unclear [ 2 , 6 , 7 ]. The aim of the present study was to clarify the possible association of HH with histological findings on a group of prospectively studied symptomatic patients with NERD. Methods Fifty patients (29 men, 21 women; aged 49.9 ± 6.6 years, mean ± SD) were evaluated prospectively in our endoscopy unit for symptoms compatible with GERD, namely heartburn, acid regurgitation and belching. A standardized questionnaire was completed for each patient during an interview with an experienced gastroenterologist. Demographic details of the GERD patients were recorded, including age, sex, smoking habits, tea, coffee and alcohol consumption, and concurrent medical conditions including hypertension and diabetes mellitus. None of the patients included in this study had a current or past history of peptic ulcer disease, previous gastric surgery, antihelicobacter therapy, or use of PPIs, non-steroidal anti-inflammatory drugs, steroids or tetracycline during the previous 4 weeks. Ethics approval was obtained from the ethics committee of the University Hospital of Alexandroupolis, and patients provided signed, informed consent for their biopsy specimens to be taken. Routine endoscopy was performed in all patients by the same endoscopist using a flexible endoscope (GIF-Q145, Olympus). The distance between the esophagogastric junction and the incisor teeth was recorded. Reflux esophagitis was graded in accordance with the Los Angeles classification [ 8 ]. HH was considered present if gastric folds were assessed as extending ≥2 cm above the diaphragmatic hiatus during quiet respiration [ 2 ]. At least four biopsy specimens were taken at 3 cm above the lower esophageal sphincter with biopsy forceps (Olympus) in a criss-cross manner. In order to improve endoscopic visualization and provide biopsy orientation, 20 ml of 2% potassium iodine solution (Lugol) was applied through a "spray" catheter [ 9 - 11 ]. To obtain sufficient material and to ensure an almost vertical pinch biopsy specimen, the opened forceps were withdrawn towards the tip of the endoscope, which was bent forwards maximally, and hence the forceps were pressed vertically against the esophageal wall. Specimens were fixed in 40 mg/L formaldehyde [ 12 ]. After all the sections had been obtained, they were assessed histologically in a blinded manner (i.e. without endoscopic or clinical information). Standardized reports completed by the histopathologist comprised an evaluation of the following histological parameters: basal zone hyperplasia, papillary length, dilatation of intraepithelial blood vessels, and semi-quantitative cellular infiltration by T-lymphocytes, neutrophils and eosinophils. Alterations in glycogen content, erosion, ulceration and chronic inflammation were also assessed as described previously [ 12 - 17 ]. The chi-square test with Yates' correlation was used to compare discrete parameters between groups. However, Fisher's exact probability test was used where expected frequencies were lower than 5. Wilcoxon's test for unpaired samples was preferred in cases of semi-quantitative parameters due to its greater power. Mean values and their 95% confidence limits were calculated. Statistical significance was set at P ≤ 0.05. All analyses were performed using the statistical software package "Statistica (version 6)". Results Endoscopy findings Endoscopy revealed esophageal mucosa with a normal appearance in 29 patients. The remaining 21 patients had esophagitis of variable severity (Table 1 ). Table 1 Endoscopy findings in patients with reflux disease. Endoscopy findings in patients with reflux disease, for HH+ and HH-. NERD ERD grade A ERD grade B ERD grade C ERD grade D Total HH+ 7 5 4 2 0 18 HH- 22 8 2 0 0 32 Total 29 13 6 2 0 50 HH was observed in 18 patients. Its presence (HH+) was correlated not only with the presence of erosions ( P = 0.0196) (Figure 1 ), but also with the severity of the endoscopy findings (Wilcoxon's T 1 score for unpaired samples: 576 for N 1 = 18 and N 2 = 32, 0.01 < P < 0.05) (Figure 2 ). Figure 1 Prevalence of HH among ERD and NERD patients. Prevalence of HH among ERD and NERD patients. P = 0.0196 when HH+ and HH- are compared. Figure 2 Relationship between HH and endoscopy findings (0.01 < P < 0.05). Relationship between HH and endoscopy findings. 0.01 < P < 0.05 when HH+ and HH- are compared. Histological findings Histological examinations of the biopsy specimens revealed esophagitis in 46 out of 48 patients, despite the normal appearance of the esophageal mucosa in most of them. Two specimens – one from a patient with ERD with HH and one from a patient with NERD with HH – were quantitatively inadequate and thus omitted. Although the remaining histological patterns were similar between ERD and NERD in HH+ (Figure 3 ), basal zone hyperplasia and loss of glycogen accompanied HH in all cases, with the correlation being highly significant in NERD ( P = 2.61 × 10 -6 ) (Figure 4 ). Figure 3 Histological findings among ERD and NERD patients in the presence of HH. Histological findings among ERD and NERD patients in the presence of HH. Basal zone hyperplasia and loss of glycogen is a ubiquitous histological feature in both ERD and NERD with HH. No statistically significant difference was observed between ERD and NERD with HH in any of the histological findings. Figure 4 Histological findings among NERD patients with and without hernia. Histological findings among NERD patients with and without hernia. P = 2.61 × 10 -6 for basal zone hyperplasia and papillary elongation. Discussion The clinical spectrum of GERD is diverse. The disease follows a rather benign course in most patients. Indeed, it is estimated that NERD accounts for up to 70% of patients with GERD [ 1 ]. The pathophysiological mechanisms that contribute to the development of GERD include delayed gastric emptying, frequent and transient relaxation of the lower esophageal sphincter, impaired esophageal clearance of regurgitated gastric acid, and HH+ [ 2 ]. HH has recently re-emerged as an important factor in GERD [ 6 , 7 , 18 ]. It may diminish lower esophageal sphincter pressure, promote acid reflux and compromise emptying of the refluxate from the distal esophagus, prolonging acid contact with the esophageal mucosa [ 19 - 21 ], a mechanism that could explain the association of HH with more severe reflux [ 22 , 23 ]. Thus, although HH has been established as the strongest predictor of the presence and severity of esophagitis in GERD patients with esophagitis, there are no published data on the role of HH in symptomatic patients without endoscopic esophagitis. Our prospective study suggests that HH+, even in patients with an esophageal mucosa that appears normal endoscopically (NERD), indicates the existence of histological effects. Our population was characterized by similar clinical presentation, and HH was correlated not only with the presence of erosions (Figure 1 ) but also with the severity of the endoscopy findings (Figure 2 ). These results further support HH as a dominant predictive factor for erosive esophagitis, which has already been confirmed in previous studies [ 2 , 24 - 27 ]. In order to further investigate the role of HH in NERD patients, we studied the role of HH+ on the histological parameters of esophagitis. In our material, basal zone hyperplasia and loss of glycogen content was detected in all HH+ ERD patients and HH+ NERD patients (Figure 3 ). In contrast, no NERD patient without HH (HH-) exhibited similar histological abnormalities (Figure 4 ). These findings probably indicate that the development of NERD in HH+ patients is more closely related to the pathophysiology of ERD, and perhaps different from the mechanisms responsible for NERD in HH- patients. Little is known about the relationship between HH and the histological variables in non-erosive esophagitis. Our finding that basal zone hyperplasia and loss of glycogen content are more frequently prevalent in HH+ than in HH- among NERD patients as well as the fact that basal zone hyperplasia, loss of glycogen content and infiltration with T-lymphocytes are more frequent in ERD than in NERD suggests the that HH contributes directly to the development of both GERD and NERD, perhaps through decreased acid clearance. Conclusions HH+ not only appears to be a risk factor for NERD, but is also suggestive of the histological presence of microscopic GERD in symptomatic NERD patients. This finding could play an important role in the therapeutic management of NERD patients with PPIs in the future, since ERD patients respond better than NERD patients to antireflux therapy. Future studies should establish whether there is a cause-and-effect relationship between HH and response to PPIs in NERD patients. List of abbreviations HH: Hiatal hernia NERD: Non-erosive reflux disease GERD: Gastroesophageal reflux disease ERD: Erosive reflux disease HH+: Presence of hiatal hernia HH-: Absence of hiatal hernia PPI: Proton-pump inhibitors Competing interests The author(s) declare that they have no competing interests. Authors' contributions A.G. participated in the endoscopy studies and in the preparation of the manuscript. K.M. participated in the endoscopy studies and in the preparation of the manuscript. A.G. participated in the histological studies. V.P. contributed to the design of the study, performed the statistical analysis and produced the graphical presentations of the results. E.S. participated in the histopathological studies. A.P. contributed to the design of the study and critically reviewed the manuscript. N.L. contributed to the design of the study and critically reviewed the manuscript. G.M. coordinated the study. All the authors read and approved the final version of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-230X/5/2/prepub | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546187.xml |
340949 | What Causes Stuttering? | The mystery of a sometimes debilitating speech disorder is examined by cognitive neuroscientists | Stuttering, with its characteristic disruption in verbal fluency, has been known for centuries; earliest descriptions probably date back to the Biblical Moses' “slowness of speech and tongue” and his related avoidance behavior (Exodus 4, 10–13). Stuttering occurs in all cultures and ethnic groups ( Andrews et al. 1983 ; Zimmermann et al. 1983 ), although prevalence might differ. Insofar as many of the steps in how we produce language normally are still a mystery, disorders like stuttering are even more poorly understood. However, genetic and neurobiological approaches are now giving us clues to causes and better treatments. What Is Stuttering? Stuttering is a disruption in the fluency of verbal expression characterized by involuntary, audible or silent, repetitions or prolongations of sounds or syllables ( Figure 1 ). These are not readily controllable and may be accompanied by other movements and by emotions of negative nature such as fear, embarrassment, or irritation ( Wingate 1964 ). Strictly speaking, stuttering is a symptom, not a disease, but the term stuttering usually refers to both the disorder and symptom. Figure 1 Speech Waveforms and Sound Spectrograms of a Male Speaker Saying “PLoS Biology” The left column shows speech waveforms (amplitude as a function of time); the right column shows a time–frequency plot using a wavelet decomposition of these data. In the top row, speech is fluent; in the bottom row, stuttering typical repetitions occur at the “B” in “Biology.” Four repetitions can be clearly identified (arrows) in the spectrogram (lower right). Developmental stuttering evolves before puberty, usually between two and five years of age, without apparent brain damage or other known cause (“idiopathic”). It is important to distinguish between this persistent developmental stuttering (PDS), which we focus on here, and acquired stuttering. Neurogenic or acquired stuttering occurs after a definable brain damage, e.g., stroke, intracerebral hemorrhage, or head trauma. It is a rare phenomenon that has been observed after lesions in a variety of brain areas ( Grant et al. 1999 ; Ciabarra et al. 2000 ). The clinical presentation of developmental stuttering differs from acquired stuttering in that it is particularly prominent at the beginning of a word or a phrase, in long or meaningful words, or syntactically complex utterances ( Karniol 1995 ; Natke et al. 2002 ), and the associated anxiety and secondary symptoms are more pronounced ( Ringo and Dietrich 1995 ). Moreover, at repeated readings, stuttering frequency tends to decline (adaptation) and to occur at the same syllables as before (consistency). Nonetheless, the distinction between both types of stuttering is not strict. In children with perinatal or other brain damage, stuttering is more frequent than in age-matched controls, and both types of stuttering may overlap ( Andrews et al. 1983 ). Who Is Affected? PDS is a very frequent disorder, with approximately 1% of the population suffering from this condition. An estimated 3 million people in the United States and 55 million people worldwide stutter. Prevalence is similar in all social classes. In many cases, stuttering severely impairs communication, with devastating socioeconomic consequences. However, there are also many stutterers who, despite their disorder, have become famous. For instance, Winston Churchill had to rehearse all his public speeches to perfection and even practiced answers to possible questions and criticisms to avoid stuttering. Charles Darwin also stuttered; interestingly, his grandfather Erasmus Darwin suffered from the same condition, highlighting the fact that stuttering runs in families and is likely to have a genetic basis. The incidence of PDS is about 5%, and its recovery rate is up to about 80%, resulting in a prevalence of PDS in about 1% of the adult population. As recovery is considerably more frequent in girls than in boys, the male-to-female ratio increases during childhood and adolescence to reach three or four males to every one female in adulthood. It is not clear to what extent this recovery is spontaneous or induced by early speech therapy. Also, there is no good way of predicting whether an affected child will recover ( Yairi and Ambrose 1999 ). The presence of affected family members suggests a hereditary component. The concordance rate is about 70% for monozygotic twins ( Andrews et al. 1983 ; Felsenfeld et al. 2000 ), about 30% for dizygotic twins ( Andrews et al. 1983 ; Felsenfeld et al. 2000 ), and 18% for siblings of the same sex ( Andrews et al. 1983 ). Given the high recovery rate, it may well be that the group abnormalities observed in adults reflects impaired recovery rather than the causes of stuttering ( Andrews et al. 1983 ). Changing Theories Over the centuries, a variety of theories about the origin of stuttering and corresponding treatment approaches have been proposed. In ancient Greece, theories referred to dryness of the tongue. In the 19 th century, abnormalities of the speech apparatus were thought to cause stuttering. Thus, treatment was based on extensive “plastic” surgery, often leading to mutilations and additional disabilities. Other treatment options were tongue-weights or mouth prostheses ( Katz 1977 ) ( Figure 2 ). In the 20th century, stuttering was primarily thought to be a psychogenic disorder. Consequently, psychoanalytical approaches and behavioral therapy were applied to solve possible neurotic conflicts ( Plankers 1999 ). However, studies of personality traits and child–parent interactions did not detect psychological patterns consistently associated with stuttering ( Andrews et al. 1983 ). Figure 2 Two Different Apparatuses to Prevent Stuttering On the left is a device by Gardner from 1899 to artificially add weight to the tongue (United States patent number 625,879). On the right is a more complex speech apparatus by Peate from 1912 (United States patent number 1,030,964). Other theories regard stuttering as a learned behavior resulting from disadvantageous external, usually parental, reactions to normal childhood dysfluencies ( Johnson 1955 ). While this model has failed to explain the core symptoms of stuttering ( Zimmermann et al. 1983 ), it may well explain secondary symptoms ( Andrews et al. 1983 ), and guided early parental intervention may prevent persistence into adulthood ( Onslow et al. 2001 ). The severity of PDS is clearly modulated by arousal, nervousness, and other factors ( Andrews et al. 1983 ). This has led to a two-factor model of PDS. The first factor is believed to cause the disorder and is most likely a structural or functional central nervous system (CNS) abnormality, whereas the second factor reinforces the first one, especially through avoidance learning. However, one should be careful to call the latter factor “psychogenic” or “psychological,” because neuroscience has shown that learning is not simply “psychogenic” but leads to measurable changes in the brain ( Kandel and O'Dell 1992 ). In some cases, arousal actually improves stuttering instead of making it worse. Consequently, some famous stutterers have “treated” their stuttering by putting themselves on the spot. Anecdotally, the American actor Bruce Willis, who began stuttering at the age of eight, joined a drama club in high school and his stuttering vanished in front of an audience. Is Stuttering a Sensory, Motor, or Cognitive Disorder? Stuttering subjects as a group differ from fluent control groups by showing, on average, slightly lower intelligence scores on both verbal and nonverbal tasks and by delays in speech development ( Andrews et al. 1983 ; Paden et al. 1999 ). However, decreased intelligence scores need to be interpreted carefully, as stutterers show a schooling disadvantage of several months ( Andrews et al. 1983 ). Associated symptoms comprise delays in tasks requiring a vocal response ( Peters et al. 1989 ) and in complex bimanual timed tasks such as inserting a string in the eye of a needle ( Vaughn and Webster 1989 ), whereas many other studies on sensory–motor reaction times yielded inconsistent results ( Andrews et al. 1983 ). Alterations of auditory feedback (e.g., delayed auditory feedback, frequency-altered feedback), various forms of other auditory stimulation (e.g., chorus reading), and alteration of speech rhythm (e.g., syllable-timed speech) yield a prompt and marked reduction of stuttering frequency, which has raised suspicions of impaired auditory processing or rhythmic pacemaking in stuttering subjects ( Lee 1951 ; Brady and Berson 1975 ; Hall and Jerger 1978 ; Salmelin et al. 1998 ). Other groups have also reported discoordinated and delayed onset of complex articulation patterns in stuttering subjects ( Caruso et al. 1988 ; van Lieshout et al. 1993 ). The assumption that stuttering might be a form of dystonia—involuntary muscle contractions produced by the CNS—specific to language production ( Kiziltan and Akalin 1996 ) was not supported by a study on motor cortex excitability ( Sommer et al. 2003 ). Neurochemistry, however, may link stuttering with disorders of a network of structures involved in the control of movement, the basal ganglia. An increase of the neurotransmitter dopamine has been associated with movement disorders such as Tourette syndrome ( Comings et al. 1996 ; Abwender et al. 1998 ), which is a neurological disorder characterized by repeated and involuntary body movements and vocal sounds (motor and vocal tics). Accordingly, like Tourette syndrome, stuttering improves with antidopaminergic medication, e.g., neuroleptics such as haloperidol, risperidone, and olanzapine ( Brady 1991 ; Lavid et al. 1999 ; Maguire et al. 2000 ), and anecdotal reports suggest that it is accentuated or appears under treatment with dopaminergic medication ( Koller 1983 ; Anderson et al. 1999 ; Shahed and Jankovic 2001 ). Hence, a hyperactivity of the dopaminergic neurotransmitter system has been hypothesized to contribute to stuttering ( Wu et al. 1995 ). Although dopamine antagonists have a positive effect on stuttering, they all have side effects that have prevented them from being a first line treatment of stuttering. Lessons from Imaging the Brain Given reports on acquired stuttering after brain trauma ( Grant et al. 1999 ; Ciabarra et al. 2000 ), one might think that a lesion analysis (i.e., asking the question where do all lesions that lead to stuttering overlap) could help to find the location of an abnormality linked to stuttering. Unfortunately, lesions leading to stuttering are widespread and do not seem to follow an overlapping pattern. Even the contrary has been observed, a thalamic stroke after which stuttering was “cured” in a patient ( Muroi et al. 1999 ). In fluent speakers, the left language-dominant brain hemisphere is most active during speech and language tasks. However, early studies on EEG lateralization already strongly suggested abnormal hemispheric dominance ( Moore and Haynes 1980 ) in stutterers. With the advent of other noninvasive brain imaging techniques like positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), it became possible to visualize brain activity of stutterers and compare these patterns to fluent controls. Following prominent theories that linked stuttering with an imbalance of hemispherical asymmetry ( Travis 1978 ; Moore and Haynes 1980 ), an important PET study ( Fox et al. 1996 ) reported increased activation in the right hemisphere in a language task in developmental stutterers. Another PET study ( Braun et al. 1997 ) confirmed this result, but added an important detail to the previous study: Braun and colleagues found that activity in the left hemisphere was more active during the production of stuttered speech, whereas activation of the right hemisphere was more correlated with fluent speech. Thus, the authors concluded that the primary dysfunction is located in the left hemisphere and that the hyperactivation of the right hemisphere might not be the cause of stuttering, but rather a compensatory process. A similar compensatory process has been observed after stroke and aphasia, where an intact right hemisphere can at least partially compensate for a loss of function ( Weiller et al. 1995 ). Right hemisphere hyperactivation during fluent speech has been more recently confirmed with fMRI ( Neumann et al. 2003 ). PET and fMRI have high spatial resolution, but because they only indirectly index brain activity through blood flow, their temporal resolution is rather limited. Magnetoencephalography (MEG) is the method of choice to investigate fine-grained temporal sequence of brain activity. Consequently, MEG was used to investigate stutterers and fluent controls reading single words ( Salmelin et al. 2000 ). Importantly, stutterers were reported to have read most single words fluently. Nevertheless, the data showed a clear-cut difference between stutterers and controls. Whereas fluent controls activated left frontal brain areas involved in language planning before central areas involved in speech execution, this pattern was absent, even reversed, in stutterers. This was the first study to directly show a neuronal correlate of a hypothesized speech timing disorder in stutterers ( Van Riper 1982 ). Thus, functional neuroimaging studies have revealed two important facts: (i) in stutterers, the right hemisphere seems to be hyperactive, and (ii) a timing problem seems to exist between the left frontal and the left central cortex. The latter observation also fits various observations that have shown that stutterers have slight abnormalities in complex coordination tasks, suggesting that the underlying problem is located around motor and associated premotor brain areas. Are there structural abnormalities that parallel the functional abnormalities? The first anatomical study to investigate this question used high-resolution MR scans and found abnormalities of speech–language areas (Broca's and Wernicke's area) ( Foundas et al. 2001 ). In addition, these researchers reported abnormalities in the gyrification pattern. Gyrification is a complex developmental procedure, and abnormalities in this process are an indicator of a developmental disorder. Another recent study investigated the hypothesis that impaired cortical connectivity might underlie timing disturbances between frontal and central brain regions observed in MEG studies ( Figure 3 ). Using a new MRI technique, diffusion tensor imaging (DTI), that allows the assessment of white matter ultrastructure, investigators saw an area of decreased white matter tract coherence in the Rolandic operculum ( Sommer et al. 2002 ). This structure is adjacent to the primary motor representation of tongue, larynx, and pharynx ( Martin et al. 2001 ) and the inferior arcuate fascicle linking temporal and frontal language areas, which both form a temporofrontal language system involved in word perception and production ( Price et al. 1996 ). It is thus conceivable that disturbed signal transmission through fibers passing the left Rolandic operculum impairs the fast sensorimotor integration necessary for fluent speech production. This theory also explains why the normal temporal pattern of activation between premotor and motor cortex is disturbed ( Salmelin et al. 2000 ) and why, as a consequence, the right hemisphere language areas try to compensate for this deficit ( Fox et al. 1996 ). Figure 3 Decreased Fiber Coherence Decreased fiber coherences, as observed with DTI, in persistent developmental stutterers compared with a fluent control group. A red dot indicates the peak difference in a coronal (top left), axial (top right), and a sagittal (bottom) slice. These new data also provide a theory to explain the mechanism of common fluency-inducing maneuvers like chorus reading, singing, and metronome reading that reduce stuttering instantaneously. All these procedures involve an external signal (i.e., other readers in chorus reading, the music in singing, and the metronome itself). All these external signals feed into the “speech production system” through the auditory cortex. It is thus possible that this external trigger signal reaches speech-producing central brain areas by circumventing the frontocentral disconnection and is able to resynchronize frontocentral decorrelated activity. In simple terms, these external cues can be seen as an external “pacemaker.” Future Directions in Research There are numerous outstanding issues in stuttering. If structural changes in the brain cause PDS, the key question is when this lesion appears. Although symptoms are somewhat different, it would be interesting to find out to what extent transient stuttering (which occurs in 3%–5% in childhood) is linked to PDS. It is possible that all children who show signs of stuttering develop a structural abnormality during development, but this is transient in those who become fluent speakers. If this is the case, it is even more important that therapy starts as early as possible if it is to have most impact. This question can now be answered with current methodology, i.e., noninvasive brain imaging using MRI. Given that boys are about four times less likely to recover from stuttering than girls, it is tempting to speculate that all stutterers have a slight abnormality, but only those that can use the right hemisphere for language can develop into fluent speakers. Language lateralization is less pronounced in women ( McGlone 1980 ) and might therefore be related to the fact that women show an overall lower incidence in PDS. Again, a developmental study comparing children who stutter with fluent controls and, most importantly, longitudinal studies on these children should be able to answer these questions. It is unlikely that stuttering is inherited in a simple fashion. Currently, a multifactorial model for genetic transmission is most likely. Moreover, it is unclear whether a certain genotype leads to stuttering or only represents a risk factor and that other environmental factors are necessary to develop PDS. Again, this question might be answered in the near future, as the National Institutes of Health has recently completed the data collection phase of a large stuttering sample for genetic linkage analysis. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC340949.xml |
503395 | Gene deletion of P-Selectin and ICAM-1 does not inhibit neutrophil infiltration into peritoneal cavity following cecal ligation-puncture | Background Neutrophil infiltration is one of the critical cellular components of an inflammatory response during peritonitis. The adhesion molecules, P-selectin and intercellular adhesion molecule (ICAM)-1, mediate neutrophil-endothelial cell interactions and the subsequent neutrophil transendothelial migration during the inflammatory response. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy, suggesting that the length of injury might be a critical factor in neutrophil infiltration. Therefore, the objective of this study was to determine the role of P-selectin and ICAM-1 in neutrophil infiltration into the peritoneal cavity during early and late phases of peritonitis. Methods Peritonitis was induced in both male wild-type and P-selectin/ICAM-1 double deficient (P/I null) mice by cecal ligation-puncture (CLP). Peripheral blood and peritoneal lavage were collected at 6 and 24 hours after CLP. The total leukocyte and neutrophil contents were determined, and neutrophils were identified with the aid of in situ immunohistochemical staining. Comparisons between groups were made by applying ANOVA and student t-test analysis. Results CLP induced a severe inflammatory response associated with a significant leukopenia in both wild-type and P/I null mice. Additionally, CLP caused a significant neutrophil infiltration into the peritoneal cavity that was detected in both groups of mice. However, neutrophil infiltration in the P/I null mice at 6 hours of CLP was significantly lower than the corresponding wild-type mice, which reached a similar magnitude at 24 hours of CLP. In contrast, in peritonitis induced by intraperitoneal inoculation of 2% glycogen, no significant difference in neutrophil infiltration was observed between the P/I null and wild-type mice at 6 hours of peritonitis. Conclusions The data suggest that alternative adhesion pathway(s) independent of P-selectin and ICAM-1 can participate in neutrophil migration during peritonitis and that the mode of stimuli and duration of the injury modulate the neutrophil infiltration. | Background Sepsis is a common cause of morbidity and mortality following surgery or trauma, and is characterized by activation of a systemic inflammatory response, severe hypotension, and major damage to multiple organs [ 1 ]. Although neutrophil migration into the tissue sites is crucial for effective elimination of infection, it also plays an important role in inflammatory tissue injury. The selectins, β2 integrins (i.e., CD18: Mac-1, LFA-1) and members of the immunoglobulin gene superfamily adhesion molecules, such as ICAM-1, play a significant role in neutrophil adhesion and transendothelial migration [ 2 - 4 ]. The expression and activation of these adhesion molecules on neutrophils and the endothelium, as well as the presence of a chemotactic gradient (eg. chemokines) appear to be important factors in neutrophil transmigration [ 3 ]. The selectins mediate neutrophil rolling while the β-2 integrins are important for firm adhesion and transendothelial migration [ 2 - 5 ]. The selectin family consists of three closely related cell surface molecules with differential expression by leukocytes (L-selectin), platelets (P-selectin), and vascular e ndothelium (P-selectin and E-selectin) [ 6 ]. ICAM-1 is one of the major ligands that binds to β-2 integrins (i.e., Mac-1 LFA-1) and is involved in neutrophil firm adhesion to endothelial and transendothelial migration [ 3 ]. ICAM-1 is constitutively expressed at a low concentration; however, under inflammatory conditions it is highly inducible in many cell types [ 7 ]. In vivo studies have shown that administration of small molecule ligands, and/or neutralizing antibodies to the selectins, ICAM-1 or β2-integrins can protect tissues from injury following endotoxin exposure, bacterial infection, or ischemia [ 8 - 10 ]. However, blocking reagents have the potential to stimulate or inhibit other receptors thus confounding the results. For example, a study by Kyriakides et al . has shown that soluble P-selectin attenuated skeletal muscle reperfusion injury by inhibition of the classical complement pathway [ 11 ]. Genetically altered mice deficient in adhesion molecules have been developed, which provide an alternative approach to study the role of the adhesion molecules in neutrophil recruitment and tissue injury. Recent studies using the transgenic mice have shown results indicating that neutrophils can use different adhesion pathways to emigrate from the systemic vasculature into the tissues and that the inflammatory responses may be site specific and stimulus dependent. For example, Mizgerd et al . have demonstrated a significant reduction in neutrophil infiltration into the peritoneal cavity at 4 hours of streptoccocal peritonitis in ICAM-1 mutant mice [ 8 ]. Another study by Kamochi et al . has shown that P-selectin and ICAM-1 significantly contributed to liver and lung injury at 4 hours of systemic endotoxemia in ICAM-1 and P-selectin/ICAM-1 double mutant mice [ 12 ]. Further, Bullard et al . have shown that P-selectin and ICAM-1 double mutant mice exhibited complete loss of neutrophil migration into the peritoneum during S. pneumoniae -induced peritonitis [ 13 ]. In contrast to these studies, Serman et al . have shown no differences in survival between wild type and ICAM-1-deficient mice following an intra-peritoneal injection with E. coli , S. auerus , or P. aeruginosa [ 14 ]. More importantly, recent clinical trials of anti-adhesion therapy in an attempt to reduce injury associated with traumatic shock and reperfusion injury failed to show a significant benefit despite strong preclinical data [ 15 ]. In an attempt to understand the disparity between the preclinical and clinical trial studies, it was noted that the lengths of injury in the clinical setting were longer than those of the preclinical studies. It appears that the underlying mechanism of neutrophil infiltration with a short period of insult is different from those of injury associated with a longer period of insult [ 15 ]. Additionally, adhesion-dependent and -independent neutrophil activation and migration can differentially be regulated by target tissue and mode of stimuli. Therefore, the goal of the study presented here was to investigate the role of ICAM-1 and P-selectin in peritonitis induced by CLP under a short and longer period of injury. The model of CLP-induced peritonitis used in this study is a clinically relevant model of sepsis [ 16 ]. This model was chosen to simulate the critical polymicrobial bacteremia-induced tissue injury that occurs in septic patients. The data of this study suggest that neutrophil infiltration into the peritoneal cavity following CLP can utilize an ICAM-1 and P-selectin independent pathway. Methods All chemicals were purchased from Sigma Chemical (St. Louis, MO), unless otherwise noted. Animals Only adult male mice (i.e., 8–10 wk) were used in this study. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1985). Experimental protocols were reviewed and approved by the Michigan State University Animal Use and Care Committee. Gene-targeted double mutant mice deficient in P-selectin and ICAM-1 (P/I double mutant), C57BL/6-Icam1 tm1Bay Selp tm1Bay , backcrossed to C57BL/6, were used in this study. Breeding pairs of double-knockout mice were purchased directly from Jackson Laboratory (Bar Harbor, ME) and bred under the guidance of University Laboratory Animal Resources at Michigan State University. The wild-type (WT) mice were male C57BL/6, which were acclimated to the animal laboratory environment for one week before the start of experimentation. Before and after surgery, all the animals had unlimited access to food and water. Induction of peritonitis Experimental model of polymicrobial sepsis Polymicrobial sepsis was induced by CLP as previously described by Chaudry, et al . [ 16 ]. Surgical utensils were sterilized and all experimental procedures were performed under aseptic conditions. Adult male mice weighing 23 to 28 gm were anesthetized with inhaled methoxyflurane (Baxter Caribe Inc., Guayama, PR). The abdominal hair was shaved, the skin was cleaned with 75% ethanol using a sterile gauze and scrubbed with betadine solution. After drying, a 2-cm midline incision was made, the cecum was identified and ligated below the ileocecal valve using 0–5 silk suture with care being taken not to occlude the cecal valves. The cecum was punctured on both sides with a 21 G needle and gently squeezed to extrude a small amount of fecal material. The cecum was then restored to its normal position and the abdomen was closed in two layers using 5.0 nylon suture. Sham animals underwent the same procedures excluding ligation and puncture of the cecum. Experimental model of peritonitis induced by 2% glycogen Adult male mice (i.e., 8–10 wk) were lightly anesthetized by inhalation anesthesia with methoxyflurane. A volume of 2% sterile glycogen solution, equal to 10% of body weight, was injected intraperitoneally (i.p.). Six hours after glycogen inoculation, mice were euthanized and peritoneal lavage and blood samples were collected. The collected samples were identified with ID numbers to assure a blind fashion performance of the tests and data analysis. Total leukocyte and differential counts were performed as described below. Peripheral blood and tissue procurement Blood samples were obtained from the right ventricle via a left anterior thoracotomy at the time of sacrifice, using a sterile heparinized syringe containing 50 μl of heparin (100 USP Units/ml). Blood smears were prepared and stained with Wright-stain (LeukoStat, Fisher Scientific, Pittsburgh, PA) for differential cell counts. The total number of peripheral blood leukocytes was determined by lysing the red blood cells using 3% acetic acid solution with the aid of a Neubauer hemocytometer. The remaining blood was centrifuged, and plasma was collected and stored at -70°C. A portion of the liver was fixed in buffered 10% formalin and embedded in paraffin, and a second portion was snap frozen in liquid nitrogen and stored at -70°C until used for immunohistochemistry staining. Collection of peritoneal lavage fluid The peritoneal fluids were collected using repetitive (2 times) instillation and withdrawal of 2 and 5 ml respectively of sterile saline solution using a syringe with a 22 G needle. The peritoneal lavage sample was placed on ice, immediately processed for centrifugation at 4°C, and the supernatant and the cell pellet were collected separately. The cell pellet was used for total and differential cell counts. Differential cell counts were determined on cytospin preparations of peritoneal lavage stained with Wright-stain (LeukoStat). The total number of peritoneal leukocytes was determined as described above for the peripheral blood leukocyte count. Additionally, the presence of neutrophils in peritoneal lavage was confirmed by immunohistochemical staining of cytospin preparation using a primary antibody (IgG2a) specific to mouse neutrophil as described below. Further, the neutrophil content was quantified by measuring the myeloperoxidase (MPO) level of peritoneal lavage as described below. Demonstration of neutrophil recruitment by myeloperoxidase assay The MPO contents of the peritoneal lavage supernatant and the cell pellet were quantified as previously published by our laboratory [ 17 ]. Briefly, the peritoneal cell pellet was resuspended in a potassium phosphate buffer, froze at -70°C, thawed, and sonicated for 40 seconds for two cycles (Ultrasonic Convertor, Model CL4, Misonix, Farmingdale, NY). The sample was then incubated at 60°C for 2 hours followed by centrifugation at 10,000 rpm for 5 minutes at 4°C. The supernatant was collected and used for the MPO assay. The MPO activity was determined using a tetramethylbenzidine substrate kit (ImmunoPure, Pierce, Rockford, IL) and read at 450 nm using a human leukocyte MPO as the standard. One unit of MPO activity was defined as the quantity of enzyme degrading 1 μmol peroxide/minute at 25°C. Similarly, the MPO content of peritoneal supernatant was measured. Determination of neutrophil infiltration and ICAM-1 expression by Immunohistochemistry To confirm the identity of neutrophils in peritoneal lavage, immunohistochemical staining was performed using acetone-fixed cytospin cell preps. The primary antibody (clone 7/4, IgG2a) specific to mouse neutrophil (Cedarlane, Westbury, NY), the biotin-conjugated secondary antibody (PharMingen, San Diego, CA), and a Vectastain avidin-biotin complex reagent and 3,3'-diaminobenzidine chromogen kits (Vector Laboratories, Inc., Burlingame, CA) were used as previously described in detail [ 17 ]. The expression of ICAM-1 on endothelial cells was examined using acetone-fixed cryosections of the tissue. The liver was used in this study. Similarly, ICAM-1 expression was identified using a specific monoclonal antibody to mouse ICAM-1 (3E 2 clone, PharmMingen, San Diego, CA) and a biotin-conjugated secondary antibody. Tissue sections were counter stained with hematoxylin (Gill's formula, Vector Laboratories) and mounted with DAKO Mounting Media (DAKO Corp, Carpinteria, CA). The samples were examined using a Nikon light microscope interfaced with a spot 24-Bit Digital Color Camera. Statistical analysis All data were expressed as means ± standard error of the mean. Comparisons between two groups were performed using an unpaired t -test by way of StatView version 5.0.1 software© for Windows. Comparisons between multiple groups and various time points were analyzed using ANOVA with subsequent Fisher's PLSD test. P ≤ 0.05 was considered significant. Results Verification of ICAM-1 and P-selectin deficiency in P/I null mice The Jackson Laboratory, where the P/I null breeding pairs were purchased, had initially tested the double knockout of the P/I null mice. In addition, the ICAM-1/P-selectin deficiency was confirmed in our laboratory in randomly selected litter mice tissue samples using RT-PCR and immunohistochemical staining of the liver tissue, as previously published by our laboratory (18). The ICAM-1 expression was determined in all the animals used in this study. Figure 1 shows ICAM-1 expression in liver tissues from the wild-type and P/I null mice by immunohistochemical staining technique. The ICAM-1 expression was constitutively present in wild-type control mice as indicated by light brown staining along the endothelium of the central vein, sinusoids, and portal vasculature (Figure 1 , WT CT). The ICAM-1 expression was markedly increased in wild-type mice following CLP and continued to be evident at 24 hours of CLP (Figure 1 , WT 6 h and WT 24 h). In contrast, ICAM-1 expression was absent in the tissues of P/I null mice before and after CLP treatment (Figure 1 , P/I CT, 6 h and 24 h). The intestinal tissue ICAM-1 expression has also been examined, which was similar to that of the liver tissue. However, liver, due to its large endothelial cell content, serves as an excellent tissue source for ICAM-1 expression. For this reason, liver is routinely used in our studies to confirm the ICAM-1 expression. Figure 1 Immunostaining of ICAM-1 expression in WT and P/I null mice. Tissues ICAM-1 expression was determined by staining the liver sections with an anti-ICAM-1 antibody specific to mouse by applying the immunoperoxidase technique, and examined under a light NIKON microscope. The top row represents wild-type (WT) control (CT), CLP 6 h and CLP 24 h, respectively. Note the increased intensity of ICAM-1 staining of central veins (arrow heads) and sinusoids (arrows) with progression of sepsis. In contrast, ICAM-1 expression in P/I null mice liver sections was completely absent in controls, 6 h after CLP and 24 h after CLP (lower row). Control group represents mice that were not subjected to sham or CLP experimental treatment. Clinical signs of sepsis Clinical signs of sepsis were manifested as quietness, lack of response to stimulus, absence of socializing behavior, ruffled hair coat and lack of appetite. All mice subjected to CLP displayed these signs that became significant at 24 hours of CLP, and no striking differences were observed between the wild-type and P/I null mice. Effect of sepsis on leukocyte count in wild-type and P/I null mice Peripheral blood leukocytes To establish the blood cell parameters under normal physiological conditions in wild-type and P/I null mice, peripheral blood samples were collected from randomly selected mice. In this article these mice are denoted as "Control", which were not subjected to the sham or CLP operation. The leukocyte and differential counts of peripheral blood were measured. As shown in Table 1 , the circulating leukocyte counts were not different between the wild-type and P/I null mice. However, the P/I null mice showed a significantly higher number of neutrophils and lower lymphocytes when compared to their wild-type counterparts (Table 1 ). There was no significant difference in monocyte count between the wild-type and P/I null mice. Table 1 Leukocyte and differential counts in WT and P/I null mice. Randomly selected normal mice not subjected to the sham or CLP treatment, were anesthetized and the peripheral blood and peritoneal lavage collected for leukocyte content and differential analysis. Data are expressed as mean ± SEM. Total leukocytes for peripheral blood represents the absolute number of cells X 10 6 / ml and for peritoneal lavage represent the absolute number of cells X 10 6 / lavage. The values in parenthesis denote absolute number of the cells X 10 6 . * p ≤ 0.05 absolute number of cells of the wild-type compared to respective P/I null mice. n represents the number of mice per each group. Source Total Leukocytes (× 10 6 ) Differential (%) % Neutrophils % Lymphocytes % Monocytes Peripheral blood WT ( n = 4 ) 10.4 ± 1.5 26 ± 2 (2.9 ± 0.5) * 72 ± 3 (7.2 ± 0.6) * 2 ± 1 (0.3 ± 0.1) P/I null ( n = 5 ) 10.3 ± 1.4 69 ± 7 (6.8 ± 0.9) 32 ± 6 (3.4 ± 0.5) 2 ± 1 (0.2 ± 0.1) Peritoneal Lavage WT ( n = 6 ) 3.8 ± 0.9 16 ± 1 (0.6 ± 0.2) 19 ± 3 (0.8 ± 0.3) 65 ± 5 (2.4 ± 0.7) P/I null ( n = 6 ) 3.6 ± 1.2 3 ± 1 (0.2 ± 0.1) 54 ± 5 (1.9 ± 0.4) 43 ± 6 (1.5 ± 0.7) The induction of CLP-induced peritonitis resulted in a significant leukopenia in wild-type as well as P/I null mice as compared to their control counterparts (Table 2 ). It is interesting to note that although not statistically significant, leukopenia in the P/I null mice was less severe than those of the corresponding wild-type counterparts. This difference was mainly reflected by the presence of a higher number of neutrophils in the P/I null mice blood (Table 2 ). Similarly, CLP induced a significant neutropenia in both wild-type and P/I null mice when compared to the respective control mice. The most severe neutropenia occurred in the wild-type mice at 24 hours of CLP. In our studies peritonitis was also induced in response to 2% glycogen, which served as a positive control stimuli to induce an acute peritonitis as have previously been reported by many other investigators. As shown in Table 2 , both wild-type and P/I null mice presented significant leukopenia at 6 hours of i.p. inoculation of 2% glycogen. Further, the inflammatory response to the sham operation, which elicited a trauma-induced peritonitis, caused leukopenia in both wild-type and P/I null mice. As shown in Table 2 , there was a significant drop in the circulating blood leukocytes at 6 and 24 hours after the sham operation when compared to the respective control mice. However, a significant neutropenia was only present at 24 hours of the sham operation. Table 2 Peripheral blood leukocyte and neutrophil counts in WT and P/I null mice subjected to CLP and 2% glycogen-induced peritonitis. Control group represents normal mice that were not subjected to sham or peritonitis treatment. Sham (CLP) represents the CLP respective sham group. Data are expressed as mean ± SEM, representing the absolute number of cells X 10 6 . * p ≤ 0.05 compared to respective control group; # p ≤ 0.05 wild-type compared to P/I null mice at the same time point and treatment. n represents the number of mice per each group. Experimental design WT mice Total Leukocytes (× 10 6 /mL) P/I null mice Total Leukocytes (× 10 6 /mL) WT mice Neutrophils (× 10 6 /mL) P/I null mice Neutrophils (× 10 6 /mL) Control ( n = 4 ) 10.1 ± 1.3 10.3 ± 2.1 2.8 ± 0.7 # 6.7 ± 1.3 Sham (CLP) 6 hr ( n = 4 ) 3.6 ± 0.6 * 4.5 ± 0.3 * 2.2 ± 0.1 # 3.8 ± 0.5 Sham (CLP) 24 hr ( n = 6 ) 2.9 ± 0.7 * 4.4 ± 0.8 * 1.4 ± 0.3 * 2.3 ± 0.1 * CLP 6 hr ( n = 6 ) 2.0 ± 0.4 * 3.4 ± 0.9 * 1.2 ± 0.3 * 2.8 ± 0.9 * CLP 24 hr ( n = 6 ) 1.1 ± 0.3 * 3.8 ± 1.1 * 0.6 ± 0.2 * 2.8 ± 0.8 * 2% Glycogen 6 hr ( n = 4 ) 3.4 ± 0.1 * 4.4 ± 0.8 * 2.5 ± 0.1 3.6± 0.6 Peritoneal leukocytes Similar to peripheral blood, the cell parameters of peritoneal fluid under normal physiological conditions in the wild-type and P/I null mice were determined in randomly selected mice. The mice were not subjected to the sham or CLP operation. As Table 1 shows, no significant difference was found in total peritoneal leukocyte counts between the wild-type and P/I null groups (i.e., WT = 3.8 ± 0.9 × 10 6 vs. P/I = 3.6 ± 1.2 × 10 6 ). However, it is interesting to note that monocytes/macrophages were the predominant cell type present in the peritoneal cavities of the wild-type mice, whereas in the P/I null mice lymphocytes were the predominant cell type (Table 1 ). Neutrophils constituted a smaller percentage of the peritoneal leukocytes in both the wild-type and P/I null mice (Table 1 ). Although the P/I null mice peritoneal lavage presented a lower percentage of neutrophils than those of the wild-type counterparts, the differences between the absolute numbers of neutrophil counts did not reach statistically significance (i.e., p = 0.08). Sepsis induced by CLP (i.e., 6 and 24 hr) caused a significant leukocyte infiltration into the peritoneal cavities of both wild-type and P/I null mice when compared to their corresponding sham group (Figure 2A ). The peritoneal leukocyte influx consisted predominantly of neutrophils as identified by Wright staining as well as in situ immunohistochemical staining using specific monoclonal antibody to mouse neutrophil (Figure 3 ). As shown in Figure 2B , a significantly greater number of neutrophils infiltrated into the peritoneal cavities of wild-type mice than those of the P/I null mice at 6 hours after CLP, which reached to comparable levels at 24 hours of CLP. Although a fewer number of neutrophils were present in the peritoneal cavities of P/I null mice, the ratio of neutrophil infiltration (i.e. ratio = infiltrated peritoneal neutrophils in response to CLP/neutrophils normally present in peritoneal cavity of the control mouse) was significantly higher in the P/I null mice than those of wild-type mice. There was a 16-fold and a 33-fold increase in the ratio of peritoneal neutrophils in wild-type at 6 and 24 hours after CLP, respectively. However, in P/I null mice, the peritoneal neutrophil infiltration ratio increased 54-fold and 204-fold at 6 and 24 hours after CLP, respectively. Figure 2 Total peritoneal leukocyte and neutrophil counts in WT and P/I null mice subjected to CLP or 2% glycogen-induced peritonitis. Wild-type (WT) and P/I null were subjected either to no treatment ( Control ), sham ( Sham CLP ), CLP, or 2% glycogen. The mice were then euthanized at 6, and 24 hours of treatment, peritoneal lavages were collected and analyzed for leukocyte and neutrophil contents. Data are expressed as mean ± SEM, representing the absolute number of cells X 10 6 per each lavage. Significant differences existed between sham, CLP and 2% glycogen as compared to their respective control group (* p < 0.05) , and as P/I null mice compared to respective wild-type at the same time period and treatment ( # p ≤ 0.05 ). Data from six independent experiments with total sample size of 4 mice per each control and 2% glycogen group, 6 to 8 mice per each sham and 8 to 11 mice per each CLP treatment group. Figure 3 Staining of cytospin preparations from peritoneal lavage. The top three rows show the Wright staining of the cellular components of peritoneal lavages. Cellular preps from the control (CT) mice of both WT and P/I null groups demonstrating mononuclear cells as the predominant cell types (left column: WT CT, P/I CT). Control group represents mice that were not subjected to sham or CLP experimental procedures. CLP induced a significant neutrophil infiltration (arrows) at 6 and 24 hours in both WT (top row: WT 6 h, WT 24 h) and P/I null mice (second row: P/I 6 h, P/I 24 h). Neutrophils were also the predominant cell type at 6 hours of 2% glycogen-induced peritonitis in WT and P/I null mice (third row: WT 6 h, P/I 6 h). The lower row represents immunoperoxidase staining of the cells (CLP mice) using an anti-neutrophil antibody specific to mouse, verifying the neutrophils as stained in brown color (WT 6 h, P/I 6 h). Peritonitis was also examined in response to 2% glycogen. A significant leukocyte infiltration into the peritoneal cavity occurred in both wild-type and P/I groups at 6 hours after 2% glycogen injection (Figure 2A ). Similar to the CLP response, the leukocyte influx into the peritoneal cavities in response to glycogen consisted predominantly of neutrophils (Figures 2B , 3 ). Further, sham operation caused a significant leukocyte infiltration into the peritoneal cavity of both wild-type and P/I null mice at 24 hours (Figures 2A , and 2B ). Similarly, this peritoneal leukocyte influx consisted predominantly of neutrophils. Demonstration of peritoneal neutrophil recruitment by myeloperoxidase assay To further quantify the degree of neutrophil infiltration into the peritoneal cavity, the MPO levels of both the peritoneal lavage cell pellets and supernatants were measured. Although the MPO levels of the cell pellets indicated the presence of a significant number of neutrophils in the peritoneal cavity after CLP, the MPO values did not always correlate with the absolute number of infiltrated neutrophils. The Wright's staining of cytospin preparations of the peritoneal cells demonstrated highly activated phagocytes with vacuolized cytoplasm often containing numerous intracellular bacteria and in most cases with loss of cellular integrity (Figure 3 ). Thus, it was of concern to find out whether the MPO contents of neutrophils were released into the extracellular environment due to activation and loss of cellular integrity. To accomplish this objective, the MPO levels of the peritoneal lavage supernatants were also measured. As figure 4B shows, at 6 hours of CLP, significant levels of MPO were detected in the peritoneal lavage supernatants of both P/I null mice and wild-type animals when compared to those of the control mice. At 24 hours of CLP, further increases of MPO levels were present in the peritoneal supernatants of both mice groups. It is interesting to note that P/I null mice demonstrated a higher MPO levels in their peritoneal lavage supernatants than the wild-type counterparts. This increase may reflect a greater degree of neutrophils activation and/or loss of cell membrane integrity of the P/I null mice, and thereby the release of their MPO into the supernatant. The differences were not statistically significant. Figure 4 Neutrophil infiltration as determined by MPO contents of peritoneal cell pellet and peritoneal supernatant. Wild-type (WT) and P/I null were subjected to CLP, euthanized at specific time points, the peritoneal lavages harvested, centrifuged, and the supernatant and the cell pellet were collected separately and assayed for MPO contents. (A) Peritoneal lavage cell pellet. (B) Peritoneal lavage supernatant. Note that at 6 h after CLP, although a significant neutrophil infiltration into the peritoneal cavity of the P/I null group is present, it is significantly impaired compared to the corresponding WT group. Values are expressed as the mean ± SEM. * p < 0.05 compared to respective control group. + p ≤ 0.05 P/I null mice compared to respective wild-type at the same time period of CLP. Data from 6 independent experiments with total sample size of 8 mice per each treatment. Demonstration of neutrophil recruitment by immunohistochemistry Previous studies have indicated that tissue MPO activity can be affected by other factors (19, 20). and that MPO values may not indicate the true presence of neutrophils. Additionally, recent studies have identified MPO as the cellular component of macrophages (i.e., Kupffer cells) (21), as well as histiocytes (22). Therefore, an immunohistochemical staining technique was used to confirm the presence of neutrophils in the peritoneal cavity. The cytospin preparations of the peritoneal lavages were immunostained with a specific antibody to mouse neutrophil. Figure 3 (lower row) shows the Immunostaining of the peritoneal lavages obtained from wild-type and P/I null mice, in which neutrophils are stained in brown color. Discussion Neutrophil infiltration is coordinated by the interplay of the adhesion molecules and the chemoattractants, which plays an important role in inflammatory tissue injury. Recent clinical trials of anti-adhesion therapy did not demonstrate a protective effect in trauma-induce shock despite very strong pre-clinical data (15). Further studies to clear this disparity suggested that the model systems applied and the length of injury are important factors that might modulate the underlying mechanism of neutrophil activation and migration (15). Therefore, this study was undertaken to examine the role of P-selectin and ICAM-1 molecules in the wild-type and P/I null mice subjected to a short and longer periods of peritonitis induce by CLP. The CLP model, chosen to mimic the normal course of sepsis in humans and animals, induced the classic signs of sepsis and a significant systemic inflammatory response. This response was associated with increased leukocyte infiltration into the peritoneal cavities of both the wild-type and P/I null mice. The leukocyte influx into the peritoneal cavity consisted predominantly of neutrophils (i.e., 62–80%), which significantly increased in both wild-type and P/I null mice at 6 and 24 hours of CLP. At the early phase of CLP (i.e., 6 h), the total number of neutrophils infiltrated into the peritoneal cavities of P/I null mice were significantly lower than those of the corresponding wild-type mice, which reached to a comparable level at 24 hours of CLP. In contrast to CLP-induced peritonitis, peritonitis induced by 2% glycogen exhibited no significant differences in the number of neutrophils infiltrated into the peritoneal cavities between the wild-type and those of the P/I null mice at 6 hours (Figure 2 ). The results of this study suggest differential regulation of the inflammatory response and neutrophil response by mode and length of the injury. The data of this study showed that under normal physiologic environment the number of leukocytes present in peripheral blood and peritoneal lavage was comparable in the P/I null and wild-type mice. However, the ratio of neutrophils and lymphocytes were reversed between these two groups. In peripheral blood of P/I mice, neutrophils constituted a larger percentage of the leukocytes, which were significantly higher than those of the wild-type mice. In a striking contrast to the peripheral blood, the P/I null mice had a smaller number of neutrophils in their peritoneal cavities, when compared to those of the wild-type mice. Previous reports have also noted an increase in peripheral blood neutrophils in mutant mice [ 13 ]. However, to our knowledge, the decrease in peritoneal neutrophils has not been previously reported. It appears that within the normal physiologic environment, neutrophil trafficking in the peritoneal cavity is low in the P/I null mice and that neutrophil transmigration is regulated through adhesion pathways that utilize P-selectin and ICAM-1 molecules. The reason for increased circulating peripheral blood neutrophil counts in the P/I null is not known. However, this phenomenon has also been previously reported in CD18, P-sel/ICAM-1 mutant mice, and in patients with moderate or severe leukocyte adhesion deficiency [ 13 , 23 , 24 ]. Further investigation is needed to determine the following: whether hematopoiesis of neutrophils production is enhanced in the P/I null mice; removal of the neutrophils from blood circulation is reduced/delayed; and/or the neutrophil's life span has been increased. In the present study, P-selectin/ICAM-1 appears to be partially involved in neutrophil migration into the peritoneal cavity but only during the early stages of the response to CLP-induced peritonitis. Conversely, these adhesion molecules were not required for maximal neutrophil migration after trauma (i.e. sham operation) or chemically-induced peritonitis (i.e 2% glycogen) and during the late phase of CLP (i.e, 24 hours). The data suggests that there might be a functional role for these adhesion molecules during the initial stages of the inflammatory response, and as the inflammatory process progresses, deficient adhesion mechanisms are bypassed. This notion has also been proposed in leukocyte recruitment in an in vivo experimental model of autoimmune encephalomyelitis, which is a T-cell-mediated disease. Kerfoot and Kubes have shown that during the early phase of encephalitis the leukocyte rolling was P-selectin dependent; however, with the progression of disease α4-integrin pathway was important in leukocyte rolling and adhesion [ 25 ]. Interaction of ICAM-1 expressed on the surface of vascular endothelial cells with the β 2 -integrins (eg., CD11b and CD18) expressed on neutrophils has shown to be a critical event mediating stable neutrophil adhesion and migration across the vascular endothelial barrier [ 26 - 28 ]. Although the study presented in this article suggests a non-role of ICAM-1 in neutrophil infiltration into the peritoneal cavity in response to peritonitis, it has to be noted that the P/I null mice are not a true ICAM-1 knockout. The P/I null mice may have a low level of alternatively spliced forms of ICAM-1 that could have been up-regulated on the vascular endothelium, and thereby promoting neutrophil migration [ 29 ]. Further, the lack of ICAM-1, per se , is not a critical factor that results in dysfunctional β 2 -integrin-mediated migration. Other adhesion molecule(s), ligand(s), and/or yet unknown counter-receptor(s) could mediate neutrophil infiltration. For example, ICAM-2, a ligand for β 2 -integrins, could be a potential candidate [ 32 ]. Other studies have shown a critical role of vascular cell adhesion molecule-1 (VCAM-1) in mediating neutrophil transendothelial migration and inflammatory tissue injury [ 30 , 31 ]. This adhesion molecule interacts with α4-integrin (α4β1, VLA-4). There is a body of evidence that α4-integrin can mediate several steps of leukocyte recruitment cascade (i.e., rolling and adhesion) through α4-integrin/MADCAM-1 (mucosal addressin cell adhesion molecule-1) and α4-integrin/VCAM-1 pathways. Vajkoczy et al., have shown that T-cells can adhere without rolling in spinal cord microvessels via α4-integrin [ 32 ]. Neutrophils express α4-integrin, and studies by Bowden et al. have demonstrated an important role of α4-integrin/VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium [ 33 , 34 ]. Additionally, the α4-integrin/VCAM-1-dependent neutrophil adhesion under flow conditions has been shown in neutrophils isolated from critically ill septic patients [ 35 ]. Neutrophil also express CD11d/CD18 and α9-integrin, which both bind VCAM-1, and could possibly, play an important role in neutrophil extravasation at sites of inflammation [ 36 ]. The importance of α4- and α9-integrin/VCAM-1 pathways in neutrophil infiltration in CLP-induced peritonitis remains unclear. It is possible that neutrophil infiltration utilizes other secondary or tertiary adhesion pathways, and/or is facilitated by proteins that mediate the function(s) of the adhesion molecules. For example, a novel glycosylphosphatidyl inositol-anchored protein (GPI-80) that may regulate β 2 -integrin-mediated cell adhesion and motility of neutrophils has been described [ 38 ]. Additionally, other proteins are recognized to act as ligands for β 2 integrins, such as those produced during coagulation as well as complement activation and tissue factor, which could facilitate neutrophil adhesion and infiltration into the peritoneal cavity [ 39 , 40 ]. Moreover, the local concentrations of chemokines appear to be critical factors in dictating the local neutrophil recruitment in an acute inflammatory response [ 41 ]. It has been shown that chemokines and their receptors are involved in leukocytes migration not only by inducing chemotaxis but also by regulating integrins to trigger cell arrest in shear flow [ 42 ]. Contrary to several other studies that have demonstrated the functional importance of P-selectin in models of myocardial infarction and inflammatory lung and liver injury, the data presented in this study indicated that P-selectin is not essential for neutrophil migration into the peritoneal cavity [ 43 - 45 ]. In support of our data, several studies have shown that blocking of P-selectin with monoclonal antibody or deletion of P-selectin and ICAM-1 did not inhibit neutrophil infiltration and tissue injury caused by hepatic reperfusion [ 18 ], endotoxin shock (46), i.p injection of 2% glycogen [ 47 ], and intestinal inflammation [ 48 , 49 ]. In contrast to our results of the P/I null mouse study presented in this article, Bullard et al., have previously reported a complete loss of neutrophil migration into the peritoneum of P/I null mice during peritonitis induced by i.p. inoculation of Streptococcus pneumonia [ 13 ]. The difference could due to the length of the experimental setting. In the Bullard et al . study, the inflammatory response and neutrophil infiltration into the peritoneal cavity was studied at 4 hours of the induction of peritonitis, while, in our study neutrophil infiltration was evaluated at 6 and 24 hours of peritonitis. In support of this reasoning is the study reported by Mizgerd et al . who have shown a compromised neutrophil migration into the peritoneal cavity of E-/P-selectin and ICAM-1 mutant mice in response to the injection of Streptococcus pneumoniae at 4 hours after injection, with no impairment present at 24 hours of bacteria injection [ 8 ]. Additionallt, Mizgerd et al . demonstrated that ICAM-1 is not necessary for neutrophil migration during glycogen-induced peritonitis, as shown otherwise by other authors [ 9 ]. Further, the difference could due to the possibility that in Bullard's study the infiltrated neutrophils into the peritoneal cavity of P/I null mice became necrotic and disintegrated upon challenges with Streptococcus pneumonia organisms. In our study, the data of peritoneal supernatant MPO levels have demonstrated a significant level of MPO released into the peritoneal cavity in mice subjected to CLP (Figure 4B ). In Bullard's study the MPO levels of the peritoneal lavages were not measured. Further, in our study the Wright staining as well as the in situ immunohistochemical staining of the peritoneal cells clearly confirms the presence of neutrophils in the cytospin preparations (Figure 3 ). The data collectively indicate that P-selectin and ICAM-1 each may have a role in neutrophil migration during early stages of acute bacterial peritonitis, but at later stages alternative pathways are recruited to mediate neutrophil migration. Functional redundancy of adhesion molecules and the cytokine production may be sufficient to compensate for the absence of P-selectin and ICAM-1 in mediating neutrophil infiltration into peritoneal cavity in response to CLP in P/I null mice. Another important difference between the results of the study presented in this article and those of previous studies may relate to the employment of essentially different models of sepsis and inflammation. While previous studies employed hemorrhagic shock-, Streptococcus- or LPS-induced peritonitis, the present study employed polymicrobial septic peritonitis as well as chemical and trauma-induced peritonitis. Published studies have shown quantitative and qualitative differences in the inflammatory response induced by gram-positive, gram-negative, polymicrobial sepsis and purified endotoxin [ 50 - 52 ]. These differences include: the species of bacteria employed and their respective antigens, the magnitude of leukocytic response, the magnitude and kinetics of cytokine production, and finally, mortality. Thus, the collective data suggest that the model of septic insult used to induce an inflammatory response is an important consideration. One major advantage of the model used in our study is that the relative magnitude of the inflammatory response in animals subjected to CLP-induced peritonitis is similar to those observed in septic patients [ 53 , 54 ]. Conclusions The data presented in this study have shown a clear, time-dependent neutrophil migration and infiltration into peritoneal cavity in responses to peritonitis, which is independent of P-selectin and ICAM-1 adhesion molecules. The negative nature of the data presented here and the failure of the anti-adhesion clinical trials, are of great importance. These findings demand innovative alternative approaches to neutrophil transmigration in inflammatory response and suggest that targets other than these adhesion molecules need to be identified. A better understanding of the mechanisms leading to neutrophil migration is critical for the development of new therapeutic strategies for treating inflammatory disease without compromising the host's immune response mechanisms. Competing interests None declared. Authors' contribution CR participated in the technical procedures for CLP, collection of blood and tissue samples, and MPO assay. KH and HA participated in preparation of tissue sample for immunohistochemistry and special staining. KH also assisted in preparation of the manuscript. EC conceived the study, and participated in its design, coordination, and preparation of the manuscript. All authors read and approved the final manuscript. Abbreviations CLP, cecal ligation-puncture; ICAM-1, Intercellular adhesion molecule-1; mAbs, monoclonal antibodies; MPO, Myeloperoxidase; P/I null mice, P-selectin/ICAM-1-deficient mice Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC503395.xml |
535814 | β-Amyloid promotes accumulation of lipid peroxides by inhibiting CD36-mediated clearance of oxidized lipoproteins | Background Recent studies suggest that hypercholesterolemia, an established risk factor for atherosclerosis, is also a risk factor for Alzheimer's disease. The myeloid scavenger receptor CD36 binds oxidized lipoproteins that accumulate with hypercholesterolemia and mediates their clearance from the circulation and peripheral tissues. Recently, we demonstrated that CD36 also binds fibrillar β-amyloid and initiates a signaling cascade that regulates microglial recruitment and activation. As increased lipoprotein oxidation and accumulation of lipid peroxidation products have been reported in Alzheimer's disease, we investigated whether β-amyloid altered oxidized lipoprotein clearance via CD36. Methods The availability of mice genetically deficient in class A (SRAI & II) and class B (CD36) scavenger receptors has facilitated studies to discriminate their individual actions. Using primary microglia and macrophages, we assessed the impact of Aβ on: (a) cholesterol ester accumulation by GC-MS and neutral lipid staining, (b) binding, uptake and degradation of 125 I-labeled oxidized lipoproteins via CD36, SR-A and CD36/SR-A-independent pathways, (c) expression of SR-A and CD36. In addition, using mice with targeted deletions in essential kinases in the CD36-signaling cascade, we investigated whether Aβ-CD36 signaling altered metabolism of oxidized lipoproteins. Results In primary microglia and macrophages, Aβ inhibited binding, uptake and degradation of oxidized low density lipoprotein (oxLDL) in a dose-dependent manner. While untreated cells accumulated abundant cholesterol ester in the presence of oxLDL, cells treated with Aβ were devoid of cholesterol ester. Pretreatment of cells with Aβ did not affect subsequent degradation of oxidized lipoproteins, indicating that lysosomal accumulation of Aβ did not disrupt this degradation pathway. Using mice with targeted deletions of the scavenger receptors, we demonstrated that Aβ inhibited oxidized lipoprotein binding and its subsequent degradation via CD36, but not SRA, and this was independent of Aβ-CD36-signaling. Furthermore, Aβ treatment decreased CD36, but not SRA, mRNA and protein, thereby reducing cell surface expression of this oxLDL receptor. Conclusions Together, these data demonstrate that in the presence of β-amyloid, CD36-mediated clearance of oxidized lipoproteins is abrogated, which would promote the extracellular accumulation of these pro-inflammatory lipids and perpetuate lipid peroxidation. | Background Hypercholesterolemia is an established risk factor for atherosclerosis and a number of recent epidemiological studies have suggested a link between increased circulating cholesterol levels and Alzheimer's disease (AD) [ 1 ]. Lipoproteins in the serum and the central nervous system (CNS) mediate cholesterol homeostasis through the delivery and removal of cellular cholesterol. With hypercholesterolemia, these phospholipid and cholesterol rich-particles accumulate abnormally outside the arterial lumen, where they are susceptible to oxidization [ 2 ]. Lipoprotein-derived oxidation products (hydroperoxides, lysophosphatidylcholine, oxysterols and aldehydes) initiate the inflammatory response that drives atherosclerotic plaque formation in the artery wall, and these lipid peroxidation products, including malondialdehyde and 4-hydroxynonal (HNE), have also been detected in AD-affected brains [ 3 , 4 ]. AD patients have been reported to have cholesterol profiles known to be pro-atherosclerotic, including increased total serum and low-density lipoprotein (LDL) cholesterol, and increased susceptibility to lipoprotein oxidation [ 5 - 9 ]. Antibodies raised against oxidized LDL (oxLDL) demonstrate reactivity to amyloid plaques and surrounding tissue, indicating that lipid peroxidation epitopes present in oxLDL accumulate in the brains of AD patients [ 3 ]. Recently, oxidized cholesterol metabolites identified in both atherosclerotic and senile plaques have been found to accelerate β-amyloid fibril formation [ 10 ]. Together, these findings suggest that, as in atherosclerosis, the accumulation of lipoprotein oxidation products in Alzheimer's disease may contribute to chronic inflammation. Phagocyte expressed pattern recognition receptors (PRR) are the first line of defense of the innate immune system against foreign or modified proteins and lipids. Scavenger receptors are pattern recognition receptors that bind and internalize a wide range of ligands, including certain polyanions, modified forms of LDL, advanced glycation endproducts and apoptotic cells [ 11 ]. These receptors are expressed by macrophages and microglia, and are the primary clearance pathway for pro-inflammatory oxidized lipoproteins [ 12 ]. In addition to binding oxLDL, several members of the scavenger receptor A (SRA) and B (CD36, SR-B1) class recognize fibrillar β-amyloid (Aβ), which accumulates in the brain and cerebral blood vessels in AD, as well as in coronary atherosclerotic plaques [ 13 - 15 ]. While studies in Sra null mice have failed to show a role for this receptor in the pathogenesis of AD [ 16 ], it has recently been demonstrated in our lab, and others, that Aβ activates an inflammatory signaling cascade via CD36 that regulates microglial activation and recruitment in the brain [ 17 - 19 ]. In AD patients, increased CD36 expression was detected in the frontal cortex which correlated with the presence of amyloid plaques and oxidative markers, suggesting that upregulation of this scavenger receptor pathway may also promote inflammation in vivo [ 20 ]. Similar to its role in peripheral macrophages, CD36 on microglia is believed to scavenge modified proteins and oxidized phospholipids. We hypothesized that a simultaneous increase in lipoprotein oxidation and accumulation of Aβ in the brain and blood vessels in AD might compromise the ability of this scavenger receptor to effectively clear these modified host ligands. Aβ has previously been shown to reduce uptake of LDL modified by acetylation, in microglia and SRA- or SR-B1-transfected cells [ 21 ]. We have shown that CD36 binds acetylated LDL with very low affinity, indicating that these studies primarily addressed the impact of Aβ on Class A scavenger receptor activity [ 12 ]. Unlike SR-A, which binds the modified apolipoprotein B component of acetylated LDL, CD36 recognizes oxidized phospholipids within the oxidized lipoprotein particle [ 22 ]. CNS lipoproteins isolated from cerebrospinal fluid, astrocytes or microglia, contain similar amounts of phospholipid, cholesterol, and cholesteryl ester content as their serum counterparts, and a pro-oxidative environment in Alzheimer's disease is believed to accelerate the formation of lipid peroxides in these particles [ 23 ]. In this study, we assessed the impact of Aβ on the binding and degradation of oxLDL via CD36, SR-A and CD36/SR-A-independent pathways. The availability of mice genetically deficient in Sra and Cd36 has facilitated studies to discriminate the actions of these individual scavenger receptors. We show that Aβ dose-dependently inhibits oxLDL binding, lysosomal degradation and cholesterol ester accumulation in macrophages and microglia. This inhibitory effect was mediated specifically via CD36 and could be reversed by removal of extracellular Aβ, indicating that the lysosomal degradation pathway was not directly impaired. Furthermore, activation of CD36-signaling by Aβ did not mediate this inhibitory effect, as targeted inactivation of essential downstream kinases did not restore oxLDL degradation. Together, these data demonstrate that Aβ impairs the ability of CD36 to scavenge oxidized lipids by competing for receptor binding. This suggests that accumulation of Aβ in the brain and vessel wall in AD would inhibit the clearance of pro-inflammatory oxidized phospholipids and oxidized-phospholipid-containing particles such as lipoproteins, thereby promoting lipid peroxidation. Methods β-Amyloid Aβ 1-42 and reverse Aβ 42-1 ( rev Aβ) peptides were obtained from Biosource International (Camarillo, California). To induce fibril formation, Aβ 1-42 was resuspended in H 2 O at 1 mg/ml and incubated for 1 week (37°C) and fibril formation was confirmed by thioflavine S (Sigma-Aldrich Co., St. Louis, Missouri) fluorescent staining as we previously described [ 17 , 18 ]. Mice The Cd36 -/- mice were generated in our laboratory as previously described [ 17 ] and SraI/II null ( Sra -/- ) mice were generously provided from Dr. T. Kodama (University of Tokyo, Japan) [ 24 ]. Both mouse lines were backcrossed to C57BL/6 mice for 7 generations (98.6% C57BL/6) prior to intercrossing to generate mice lacking both Sra and Cd36 . Double knockout mice ( Sra -/- / Cd36 -/- ) were generated from heterozygote intercrosses at the expected ration of 1:16. Wild type age-matched C57BL/6 mice (The Jackson Laboratory, Bar Harbor, Maine) were used as controls for these three lines. Lyn -/- and Fyn -/- mice were obtained from The Jackson Laboratory and Lyn -/- , Fyn -/- and wild type littermate control mice were generated from heterozygote intercrosses. All mice were maintained in a pathogen-free facility with free access to rodent chow and water. All experimental procedures were carried out in accordance with Massachusetts General Hospital's institutional guidelines for use of laboratory animals. Primary macrophage and microglial culture Macrophages were collected from 6–8 week old mice by peritoneal lavage 4 days after i.p. injection with 3% thioglycollate as we previously described [ 17 , 25 ]. Cells were washed in PBS, cultured for 2 h in DMEM with 5% FCS, and washed again to remove non-adherent cells. Adherent cells were incubated in DMEM with 1% FCS overnight prior to use and were routinely >95% CD11b + and F4/80 + as determined by flow cytometric analysis. Primary microglia were prepared from mixed brain cultures of neonatal mice as we previously described [ 17 ]. Briefly, whole brains were incubated in 0.25% trypsin and 1 mM EDTA (10 min, 25°C) and dissociated to obtain a single cell-suspension. Cells were washed in HBSS (4x, 10 min) and cultured in DMEM containing 10% FCS, 1% Fungizone for 10–12 days. Microglia accumulating above astrocyte monolayers were collected after gentle agitation, washed and incubated in DMEM with 1% FCS overnight prior to use. Microglia prepared in this manner were routinely >95% CR3 + and express SR-A and CD36 [ 14 , 17 , 18 ]. Lipoproteins Human 125 I-LDL and LDL (d = 1.019 - 1.063) were purchased from Biomedical Technologies (Stoughton, Massachusetts) and oxidized as we previously described [ 12 , 26 ]. LDL was diluted to 250 μg/ml, dialyzed against PBS at 4°C to remove EDTA, and then dialyzed against 5 μM CuSO 4 in PBS at 37°C for 6 or 10 h. Oxidation was terminated by the addition of 50 μM butylated hydroxytoluene and 200 μM EDTA and oxLDL was used within 2 days of preparation. Moderately oxidized LDL (6 h oxidation) had a relative electrophoretic mobility of approximately 2.5–3 times that of native, unmodified LDL, whereas extensively oxidized LDL (10 h oxidation) had a relative mobility four times that of native LDL. 125 I-OxLDL degradation, binding and uptake assays Measurement of 125 I-oxLDL binding, degradation and uptake was performed on confluent monolayers of peritoneal macrophages (7 × 10 5 ) and microglia (5 × 10 5 ) in 24 well plates as we previously described [ 12 , 26 ]. Briefly, 10 μg/ml of 125 I-oxLDL was added to cells in the presence or absence of 30-fold excess unlabeled oxLDL, native LDL, Aβ 1-42 , or rev Aβ peptide for 5 h at 37°C. To measure 125 I-oxLDL degradation, media were removed and assayed for TCA-soluble non-iodide degradation products. To measure 125 I-oxLDL binding in the presence Aβ 1-42 or rev Aβ, cells were washed 3x with 50 mM Tris pH 7.4, 0.15 N NaCl and 2 mg/ml BSA, 1x with 50 mM Tris pH 7.4 and 0.15 N NaCl and treated with 0.4% dextran sulfate to release surface bound 125 I-oxLDL [ 27 ]. To measure 125 I-oxLDL uptake, cells were washed 3x in 50 mM Tris pH 7.4 and 0.15 N NaCl, lysed in 0.1 N NaOH and assayed for 125 I and cellular protein content. In some experiments, cell-association of oxLDL (cell-surface bound and endocytosed oxLDL) was measured by omitting the dextran sulfate treatment. Cellular protein content was measured by BCA assay (Pierce, Rockford, IL) and degradation, binding and uptake activity are expressed as ng 125 I-oxLDL/mg protein. Specific degradation was calculated as the difference of total cellular degradation of 125 I-oxLDL in the presence and absence of 30-fold excess unlabelled oxLDL competitor. All measurements were performed in triplicate and are representative of at least 3 experiments. Analysis of cellular cholesterol content Macrophages and microglia were cultured with 40 μg/ml of oxLDL for 48 h in the presence or absence of Aβ 1-42 or revAβ. Cholesterol ester accumulation was assessed by gas chromatography-mass spectrometry (GC-MS) and oil red O staining as we previously described [ 12 , 26 ]. For GC-MS analysis, lipids were extracted with hexane:isopropanol (3:2) and stigmasterol (Sigma, St. Louis, Missouri) was added as an internal standard. Lipid extracts were washed once with water and divided equally. One lipid aliquot was saponified for determination of total cholesterol and the second aliquot analyzed for free cholesterol using gas chromatography-mass spectrometry. The samples were injected (splitless) into an Agilent 6890 GC-MS-(G2613A system, Agilent Technologies, Palo Alto, CA) equipped with a J&W DB17 fused silica capillary column (15 m × 0.25 mm inner diameter × 0.5 μm; J&W Scientific, Folsom, CA). The GC temperature program was as follows: the initial temperature was 260°C for 5 min, then increased to 280°C (5°C/min) and held 280°C for 11 min. A model 5973N mass-selective detector (Agilent Technologies) was used in scan modes to identify the samples. Cholesterol measurements were made in triplicate and normalized to cellular protein content. Cholesterol ester content was calculated by subtracting free cholesterol from total cholesterol measured after saponification. To assess neutral lipid accumulation, cells were fixed in 4% paraformaldehyde and stained with oil red O for 30 min. Staining was recorded on an Olympus X10 microscope equipped with a digital camera. Real time RT-PCR analysis Total RNA was extracted using Trizol B reagent and real-time quantitative RT-PCR (QRT-PCR) was performed using the QuantiTect SYBR Green PCR kit (Qiagen Inc, Valencia, CA) as we previously described [ 17 , 18 ]. Each reaction contained 0.3 μM of CD36, SRA or GAPDH primers, 3 μl of cDNA, SYBR Green, and HotStarTaq polymerase. PCR was performed using a BioRad i Cycler under the following conditions: 15 min at 95°C, followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C and 30 sec at 72°C. Each sample was analyzed in triplicate and the amount of CD36, SRA and GAPDH mRNA in each sample was calculated from a standard curve of known template. Data are expressed as the mean number of CD36 and SRA molecules normalized to GAPDH. Western analysis Cells were washed in ice-cold PBS and lysed in radioimmune precipitation buffer containing protease and phosphatase inhibitors. For detection of CD36, 30 μg of protein was run on an 8% denaturing SDS-polyacrylamide gel, transferred to nitrocellulose and blocked overnight in 5% nonfat dry milk and 3% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) as we previously described [ 17 , 26 ]. Membranes were incubated with a rabbit anti-CD36 antiserum (1:500 dilution) generated in our laboratory [ 17 ] for 2 hours, washed three times in TBS-T, and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000 dilution) for 1 hour. Blots were washed 3x in TBS-T, exposed to ECL reagent (Amersham Biosciences, Piscataway, NJ), and signal was recorded and quantified using an Alpha Innotech Fluorchem 8800 image analysis system. Blots were stripped and probed with an anti-actin rabbit polyclonal antibody (Santa Cruz Biotechnology) as described above as an internal standard for equivalent loading. Results β-Amyloid blocks oxidized LDL metabolism and cellular cholesterol accumulation in macrophages and microglia Treatment of peritoneal macrophages with Aβ 1-42 , but not rev Aβ, dose-dependently inhibited lysosomal degradation of 125 I-oxLDL (Fig. 1a ). Half-maximal inhibition of macrophage 125 I-oxLDL degradation was achieved with 10 μM Aβ 1-42 . This was equivalent to the inhibitory effect of 15-fold excess of unlabelled oxLDL competitor (Fig. 1b ). At 20 μM, Aβ 1-42 reduced macrophage degradation of 125 I-oxLDL by up to 90%, while treatment with the same concentration of non-fibrillar rev Aβ peptide reduced degradation by only 10%, and this concentration was selected for all further experiments. Because engulfment of Aβ 1-42 has previously been reported to disrupt endosomal/lysosomal integrity in a neuronal cell line [ 28 ], we investigated whether the observed reduction in oxLDL degradation could be attributed to lysosomal accumulation of Aβ 1-42 which occurs within 1 h of treatment. After exposure to Aβ 1-42 for 3 hours, macrophages were washed extensively to remove extracellular Aβ 1-42 and exposed to 125 I-oxLDL or 125 I-oxLDL + Aβ 1-42 for 5 h. While cells continuously exposed to Aβ 1-42 showed a profound impairment of oxLDL degradation, cells pre-treated with Aβ 1-42 were similar to untreated and revAβ-treated cells, indicating that intracellular accumulation of Aβ 1-42 does not block subsequent lysosomal degradation of oxLDL (Fig. 1c ). Figure 1 Aβ inhibits lysosomal degradation of oxidized LDL and cholesterol ester accumulation in macrophages. A. Fibrillar Aβ, but not revAβ, dose-dependently inhibits lysosomal degradation of 125 I-oxLDL by macrophages, similar to unlabeled oxLDL competitor (B). C. Intracellular accumulation of Aβ does not block lysosomal degradation of 125 I-oxLDL. Macrophages were pretreated with 20 μM Aβ or revAβ for 3 hours to allow intracellular accumulation, washed extensively to remove extracellular peptide and degradation of 125 I-oxLDL over 5 h was measured in the absence (PT) or presence of additional peptide. D. Aβ blocks cholesterol ester accumulation in oxLDL treated macrophages. Cellular lipids were extracted from macrophages treated with oxLDL (40 μg/ml) for 48 h in the presence or absence of 20 μM Aβ and analyzed by gas-chromatography mass-spectrometry. Cholesterol ester content was normalized to cellular protein. (A-D) Data are the mean of triplicate samples ± standard deviation, *p ≤ 0.005. The inhibition of 125 I-oxLDL degradation by Aβ 1-42 would be predicted to reduce cellular cholesterol ester accumulation. Excess unesterified "free" cholesterol is cytotoxic and is thus rapidly converted by the microsomal enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) to cholesterol ester for storage. This neutral lipid is retained in cytoplasmic lipid droplets for storage and/or efflux from the cell. Using gas chromatograpy-mass spectrometry, we quantified the cholesterol ester content of macrophages treated with oxLDL in the presence and absence of Aβ 1-42 . As expected, untreated cells did not contain measurable cholesterol ester, while macrophages treated with 40 μg/ml oxLDL for 48 h accumulated approximately 80 μg cholesterol ester/mg cellular protein (Fig. 1d ). By contrast, macrophages treated with both oxLDL and Aβ 1-42 showed no measurable cholesterol ester accumulation after 48 h, similar to untreated cells. As seen in peripheral macrophages, Aβ 1-42 substantially inhibited 125 I-oxLDL binding, uptake, and degradation by primary microglia indicating that it has a similar effect on lipoprotein metabolism in these two myeloid cell types (Fig. 2a,2b,2c ). In the presence of 20 μM Aβ 1-42 , microglia demonstrated a 55% reduction in 125 I-oxLDL binding, an 80% reduction in 125 I-oxLDL uptake and a 95% reduction of 125 I-oxLDL degradation. The absence of cholesterol ester in oxLDL treated microglia exposed to Aβ 1-42 was confirmed by staining cells with the neutral lipid stain oil red O. Microglia treated with oxLDL alone demonstrate oil red O positive lipid droplets in their cytoplasm characteristic of cholesterol ester storage (Fig. 2d ). However, in the presence of Aβ 1-42 , oxLDL treated microglia show a dramatic reduction in lipid droplets that is not seen with treatment with the same concentration of rev Aβ. As expected, cells treated with Aβ 1-42 or rev Aβ alone do not accumulate cholesterol ester in the absence of exogenously added oxLDL (Fig. 2d ). Similar results were observed in macrophages (data not shown). Together, these data demonstrate that Aβ blocks cholesterol ester accumulation in macrophages and microglia by inhibiting oxLDL clearance. Figure 2 Aβ inhibits oxLDL binding, uptake and degradation in microglia. Treatment of primary microglia with 20 μM fibrillar Aβ, but not revAβ, inhibits 125 I-oxLDL binding (A), cellular uptake (B) and degradation (C). Data are the mean of triplicate samples ± standard deviation, *p ≤ 0.005. (D) Microglia treated with 20 μM fibrillar Aβ fail to accumulate cholesterol ester in the presence of oxLDL. Microglia were incubated with 40 μg/ml oxLDL for 48 h in the presence and absence of 20 μM Aβ or revAβ peptide and stained with oil red O to visualize neutral lipid. Cells treated with oxLDL alone or in the presence of revAβ demonstrate the accumulation of red-stained lipid droplets in the cytoplasm. By contrast, oil red O staining is greatly reduced in oxLDL and Aβ co-treated microglia. Mag. 200X. fAβ downregulates expression of the OxLDL receptor CD36 To address the mechanism by which Aβ 1-42 inhibits oxLDL metabolism, we first evaluated cellular expression of the scavenger receptors SRA and CD36. Fibrillar Aβ 1-42 reduced expression of CD36 mRNA by 40 and 60% after 6 and 24 h, respectively (Fig. 3a ), but showed no effect on macrophage expression of SRA. Western blotting confirmed a 40% decrease in CD36 protein in Aβ 1-42 treated macrophages (Figure 3b ), which would be expected to reduce the ability of these cells to bind oxLDL. Figure 3 Aβ downregulates expression of the oxLDL receptor CD36. A. Analysis of CD36 and SRA mRNA in peritoneal macrophages treated with Aβ (20 μM) by quantitative RT-PCR. Data represent the mean of triplicate samples ± standard deviation, *p ≤ 0.005. B. Western blot analysis confirming CD36 protein downregulation by Aβ. The signal was recorded and the integrated density value quantified using an Alpha Innotech FluorChem Imager and normalized to actin protein. Data are representative of 2 experiments. fAβ competes for oxLDL binding to CD36, but not SRA β-Amyloid has previously been reported to bind to the class A scavenger receptors SRA I & II and to block uptake of LDL modified by acetylation [ 14 , 21 ]. We employed Sra and Cd36 single null mice to investigate the role of these receptors in the inhibition of oxLDL clearance by Aβ 1-42 . In addition, we used Sra / Cd36 double null mice to evaluate the role of SRA/CD36-independent mechanisms, including those of additional scavenger receptor family members. Because of the difficulty of culturing sufficient numbers of primary microglia for binding and degradation experiments, studies involving knock-out mice were performed with peritoneal macrophages. In Sra -/- and wild type macrophages Aβ 1-42 blocked cell association (binding and uptake) of 125 I-oxLDL by greater than 50%, indicating that this scavenger receptor is not essential for the inhibitory action of Aβ (Fig. 4a ). By contrast, in the absence of Cd36 , impairment of 125 I-oxLDL cell association by Aβ 1-42 was reduced to 8%, indicating that this receptor was the primary target of Aβ 1-42 inhibition (Fig. 4a ). Figure 4 Inhibition of oxLDL cell-association by Aβ requires CD36, but not CD36-associated signal transduction. A. To determine whether SRA or CD36 was essential for Aβ-inhibition of oxLDL metabolism, cell-association of 125 I-oxLDL was measured in wild type, Sra -/- and Cd36 -/- macrophages in the presence or absence of 20 μM Aβ. While Aβ blocked oxLDL association by approximately 50% in wild type and Sra -/- macrophages, this effect was lost in Cd36 -/- macrophages indicating that CD36 is required for this inhibition. B. Inhibition of 125 I-oxLDL degradation by Aβ does not utilize the Aβ-CD36 signaling pathway involving Lyn and Fyn kinases. Aβ impaired oxLDL degradation to a similar extent in wild type, and Lyn -/- or Fyn -/- macrophages in which CD36-signaling is impaired, indicating that this signal transduction pathway is not required, Data are the mean of triplicate samples ± standard deviation, *p ≤ 0.005. The finding that CD36 is required for Aβ 1-42 inhibition of oxLDL suggests two possible mechanisms of action: (1) direct competition for CD36 binding, or (2) inhibition of oxLDL metabolism as a result of Aβ/CD36 signal transduction. To address whether CD36 signaling inhibits cellular oxLDL degradation, we used macrophages with targeted deletions in two kinases in this pathway, Lyn and Fyn, which have previously been shown to be required for CD36-mediated p44/42 activation, MCP-1 secretion and ROS production [ 17 ]. However, as in wild type macrophages, Aβ 1-42 effectively inhibited 125 I-oxLDL degradation in Lyn -/- and Fyn -/- macrophages, suggesting that this signaling pathway does not inhibit oxLDL metabolism (Fig. 4b ). Furthermore, treatment of macrophages with the general phosphotyrosine kinase inhibitor genistein did not reverse Aβ 1-42 inhibition of 125 I-oxLDL degradation, confirming that phosphotyrosine kinase signaling does not mediate this effect of Aβ 1-42 (data not shown). Interestingly, in untreated Fyn -/- macrophages 125 I-oxLDL degradation was increased 2-fold (Fig. 4b ) indicating that this kinase may play a role in regulating oxLDL uptake. However, despite a doubling of oxLDL degradation in Fyn -/- macrophages, this process was still inhibited by Aβ 1-42 by up to 90%. Together, these experiments suggest that Aβ 1-42 inhibition of oxLDL metabolism is not the result of CD36-Lyn/Fyn signal transduction and support the hypothesis that Aβ 1-42 competes for oxLDL binding to CD36. Analysis of 125 I-oxLDL cell-surface binding showed that Aβ inhibited 125 I-oxLDL binding by approximately 60% in wild type macrophages (Fig. 5 ). This inhibitory effect was lost in Cd36 -/- macrophages, confirming that Aβ inhibited oxLDL binding to this receptor. Of note, wild type macrophages bound 60% more oxLDL than macrophages lacking Cd36 as has previously been reported, and this correlated with the percentage reduction of oxLDL binding by Aβ in wild type macrophages (57%), suggesting that the CD36-dependent contribution to oxLDL binding was totally inhibited. To confirm that other myeloid scavenger receptors were not inhibited by Aβ, assesed 125 I-oxLDL binding in Sra -/- Cd36 -/- macrophages. No effect of Aβ was observed in these cells, demonstrating the specificity of Aβ inhibition of oxLDL binding to CD36. Figure 5 Inhibition of oxLDL binding requires CD36, but not other scavenger receptors. Binding of 125 I-oxLDL was measured in wild type, Cd36 -/- or Cd36/Sra -/- macrophages in the presence or absence of 20 μM Aβ to assess the role of CD36 and CD36/SRA-independent pathways. In the absence of CD36, oxLDL binding was not reduced by Aβ, indicating that this receptor is the target of Aβ inhibition. Binding of oxLDL via other scavenger receptors, which is measurable in Cd36/Sra -/- macrophages, was not inhibited by Aβ. Data are representative of triplicate samples ± standard deviation, *p ≤ 0.005. Discussion Numerous studies have demonstrated elevated markers of lipid peroxidation in the brains, CSF and plasma of Alzheimer's disease patients, including thiobarbituric acid-reactive substances, 4-hydroxy-2-nonenal (HNE), acrolein and F2-isoprostanes, which are suggestive of a persistent pro-oxidant environment [ 3 , 4 , 9 , 29 , 30 ]. Lipoprotein particles are especially vulnerable to free-radical mediated lipid peroxidation and the resulting peroxy fatty acids are highly unstable, readily decomposing to form peroxy and alkoxy radicals that further promote oxidation. This self-propagating cycle of lipid peroxidation is particularly damaging in lipid-rich tissues such as the brain, and as a result, the innate immune system has evolved mechanisms to rapidly recognize and clear oxidized lipids. The myeloid scavenger receptors are the first lines of defense against these non-native lipids, as well as modified host proteins such as β-amyloid [ 11 , 31 ]. This dual responsibility prompted us to evaluate whether macrophages and microglia would be compromised in their ability to metabolize oxidized lipoproteins in the presence of Aβ. We found that fibrillar Aβ specifically inhibited all phases of oxLDL metabolism, including binding, uptake, degradation and accumulation of cellular cholesterol ester. This was mediated by a selective inhibition of CD36 binding by Aβ, as well as a decrease in CD36 mRNA and protein expression. However, inhibition of oxLDL metabolism was independent of the recently identified Aβ-CD36-signaling cascade, as targeted inactivation of essential downstream kinases did not restore cellular oxLDL degradation. Together, these data demonstrate that oxidized lipoprotein metabolism by CD36 is profoundly impaired in the presence Aβ, and suggest that accumulation of Aβ in the brain and blood vessels in AD would foster the extracellular persistence of these pro-inflammatory lipids, thereby perpetuating lipid peroxidation. Thus, Aβ binding of CD36 in the brain would promote inflammation via two specific mechanisms: (1) through its engagement of signal transduction and microglial recruitment, and (2) through its abrogation of this important clearance pathway for oxidized phospholipid-containing ligands. In addition to CD36, two other scavenger receptor family members have been shown to be expressed in the brain and to bind Aβ. The Class A scavenger receptors, SRA I and II, and the class B SR-BI are expressed by neonatal microglia, but unlike CD36, these receptors are not expressed by microglia in the normal adult brain [ 14 , 15 ]. However, microglial expression of SRA is increased during AD, and this receptor can mediate both adherence to Aβ and its phagocytosis [ 14 , 32 , 33 ]. In Sra -/- mice, there is a 60% impairment in microglial binding of Aβ and reactive oxygen production, however, AD-associated brain pathology is not reduced [ 16 , 33 ]. SRA ligands, including acetylated LDL and fucoidan, reduce Aβ uptake by microglia, however these ligands may also affect other receptors [ 34 ]. Conversely, Aβ and its soluble precursor protein, sAPPα, inhibit macrophage and microglial uptake of acetylated LDL [ 14 , 21 , 35 ]. While acetylated LDL is not believed to occur physiologically, other modifications of LDL, such as oxidation, that allow binding to SRA may also be competed by Aβ. However, in our assays Aβ inhibition of oxLDL binding and degradation did not occur via this pathway, similar effects were seen in wild type and Sra -/- cells. By contrast, the effect of Aβ was abolished in the absence of CD36, indicating that this receptor is the target of Aβ action. The difficulty in isolating human lipoproteins from the CNS has limited their experimental use, however, several groups have shown that oxidized serum lipoproteins, including LDL, HDL and VLDL, are toxic to neurons [ 36 - 39 ], and both oxLDL and oxidized CSF lipoproteins disrupt neuronal microtubule organization, a pathogy characteristic of the AD brain [ 6 , 38 , 40 ]. Thus, the loss of CD36-mediated oxidized lipoprotein clearance in the presence of Aβ 1-42 would be predicted to foster inflammation and tissue injury. While we have shown that Aβ blocks CD36 binding of oxLDL, and its subsequent degradation, we would predict that similar results would be found with oxidized lipoproteins isolated from the CNS, astrocytes or microglia. Although serum and brain lipoprotein particles differ in their apolipoprotein composition [ 23 , 41 - 44 ], they contain similar amounts of cholesterol, cholesterol ester and phospholipid. CD36 has been shown to recognize a phospholipid moiety of oxidized lipoproteins, primarily oxidized phosphatidylcholine, which is abundant in CSF lipoproteins [ 22 , 41 ]. The presence of a pro-oxidant environment in AD would be expected to generate similar modifications of CSF lipoproteins and lipoproteins isolated from AD-affected individuals have, in fact, been shown to be more susceptible to oxidation [ 5 , 6 ]. Inhibition of the primary clearance pathway for oxidized lipoproteins would be predicted to promote inflammation and persistence of lipid peroxidation. Disruption of oxidized lipoprotein metabolism by Aβ may also be relevant in the context of atherosclerosis. Cholesterol oxidation products generated during the inflammatory component of atherosclerosis have been shown to accelerate β-amyloid fibril formation [ 10 , 45 ]. Furthermore, a recent study identified Aβ advanced human atherosclerotic plaques [ 46 ]. Our data suggests that the presence of Aβ in the artery wall may both prevent macrophage oxidized LDL uptake via CD36, thereby promoting β-amyloid fibril formation and activating CD36-signaling [ 47 ]. It has recently been shown that Aβ-CD36-signaling leads to the expression of cytokines and chemokines, including IL-1β, TNFα, MCP-1, MIP-1α and β and MIP-2 [ 17 - 19 ]. Activation of this signaling cascade would be predicted to promote inflammation, as well as atherosclerotic plaque progression. Indeed, overexpression of a mutant human amyloid β-precursor protein in an atherosclerosis-susceptible mouse strain (B6Tg2576) led to significantly increased levels of atherosclerosis, which correlated positively with cerebral Aβ deposits [ 48 ]. Of particular interest, when these mice were maintained on a normal chow diet that did not induce atherosclerosis in wild type littermates, B6Tg2576 mice developed early atherosclerotic lesions in the aortic root, suggesting that Aβ promotes atherogenesis. The convergence of risk factors for AD and atherosclerosis suggest that these chronic inflammatory diseases may have overlapping mechanisms of pathogenesis in which cholesterol levels and lipid peroxidation play a central role. List of abbreviations used Aβ, β-amyloid peptide 1–42; ACAT, acyl-coenzyme A:cholesterol acyltransferase; AD, Alzheimer's disease; CSF, cerebral spinal fluid; DMEM, Dubelcco's modified Eagle medium; FCS, fetal calf serum; fAβ, fibrillar Aβ; GC-MS, gas chromatography-mass spectrometry HNE, 4-hydroxy-2-nonenal; oxLDL, oxidized low density lipoprotein; rev Aβ, reverse β-amyloid peptide 42-1; SRA, scavenger receptor A; SR-BI, scavenger receptor B I. Competing interests The authors declare that they have no competing interests. Authors' contributions VVK performed the measurements of 125 I-oxLDL binding, uptake and degradation, and participated in the design of the study and analysis of results. LAM and TK isolated the primary microglia and macrophages, performed western blots, quantitative RT-PCR, and measurements of 125I-oxLDL binding, uptake and degradation. AAT performed measurements of 125 I-oxLDL binding, uptake and degradation. KJM conceived of the study, participated in its design and wrote the manuscript. All authors read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535814.xml |
517492 | XHM: A system for detection of potential cross hybridizations in DNA microarrays | Background Microarrays have emerged as the preferred platform for high throughput gene expression analysis. Cross-hybridization among genes with high sequence similarities can be a source of error reducing the reliability of DNA microarray results. Results We have developed a tool called XHM (cross hybridization on microarrays) for assessment of the reliability of hybridization signals by detecting potential cross-hybridizations on DNA microarrays. This is done by comparing the sequences of the probes against an extensive database representing the transcriptome of the organism in question. XHM is available online at . Conclusions Using XHM with its user-adjustable parameters will enable scientists to check their lists of differentially expressed genes from microarray experiments for potential cross-hybridizations. This provides information that may be useful in the validation of the microarray results. | Background The development of DNA microarrays has revolutionized high throughput gene expression analysis. The two main platforms are: cDNA microarrays, where PCR products of individual cDNA fragments are immobilized on glass slides [ 1 ], and oligonucleotide microarrays, where oligonucleotides in situ synthesized or spotted on glass slides are used [ 2 , 3 ]. In a typical cDNA microarray experiment, total cellular RNA from two sources, a reference (or control) and an experimental sample are converted to cDNA by reverse transcription, labeled with two different fluorescent colors, and hybridized to an array of cDNA probes. One of the major concerns of cDNA microarrays is cross-hybridization of the labeled RNA (or cDNA) to non-target homologous probe sequences on the array [ 4 , 5 ]. Cross-hybridizations that may arise due to poly(A)-tail of mRNA or repetitive elements may be reduced or eliminated using hybridization blocking reagents like poly(A) oligonucleotides [ 6 ] or Cot1 DNA. Another major source of cross-hybridization may come from conserved sequences shared by two or more cDNAs, such as gene family members [ 4 , 7 ]. Analysis of sequenced multicellular eukaryotic genomes suggests that a large percentage of genes belong to gene families, some of which have high sequence similarities [ 8 ]. A recent study reported that approximately 17% of human transcripts in the UniGene database contain perfect match repeats of 20 bp minimum lengths [ 9 ]. From this study, however, the frequency of long, non-perfect match sequences such as those shared among paralogs, was not clear. Some studies have suggested that cross-hybridization can be a source of errors in cDNA microarray experiments [ 3 , 4 , 7 , 9 , 10 ]. In general, these studies indicate that cDNAs with nucleotide sequence identities higher than 70–80% over a certain length show significant levels of cross-hybridization. Analysis of chemokines, cytochrome P450 isozymes, G proteins and protease homologous gene families showed that cross-hybridization signals of 0.6–12% and 26–57% could arise from shared nucleotide identities of 55–80% and >80%, respectively [ 4 ]. Using synthetic 50 mer oligonucleotides, it was also shown that non-target sequences with >75–80% identity to 50 mer probes in microarrays could result in cross-hybridization [ 3 ]. In addition, cross-hybridization was also observed if a non-target sequence included stretches of ≥ 15 continuous bases identical to a 50 mer probe sequence [ 3 ]. Cross-hybridization is thought to contribute to discrepancies of results observed between oligonucleotide arrays and cDNA arrays [ 11 ]. Cross-hybridization may occur for both oligonucleotide and cDNA microarrays. An advantage with oligonucleotide arrays is that the complete sequences of the probes are known. Because cDNA arrays are constructed from PCR products of cDNA clones sequenced from 3'- or 5'-ends, the complete sequences of the spotted probes may not be available. We have not been able to identify programs or web servers capable of doing flexible cross-hybridization analysis. Programs with related functionality have been described, including ProbeWiz [ 12 ], OligoWiz [ 13 ] and PROBEmer [ 14 ], but these are for designing specific probes by avoiding regions of the cDNAs where there may be a cross-hybridization problem. For the assessment of the cross-hybridization potential in DNA microarray analysis, a more specific tool is required. In the present study we limit ourselves to the following problem: Given a probe (or a set of probes), identify the targets to which it can hybridize given user-defined criteria. The exact criteria to be used to decide whether two genes with high sequence similarity can cross-hybridize will depend on a number of factors, including the set of probes used and the experimental protocols – in particular hybridization and washing stringencies. We propose to use generic forms of criteria allowing the user to choose parameters in order to define the criteria for cross-hybridization. We investigate whether BLAST can be used to detect targets satisfying a set of criteria for cross-hybridization, based on previous experimental findings [ 3 , 4 ], and assess different (transcript) databases in order to evaluate their suitability for our purpose. A web tool is developed that allows the user to query precomputed results from several probe sets, or to analyze own probes with respect to a database representing transcripts in the organism where the microarray experiments are performed. The tool is applicable to analysis of both oligonucleotide and cDNA probes. Below we describe the developed XHM (cross hybridization on microarrays) system. We also include analysis performed in order to identify appropriate analysis methods and databases. Furthermore we describe the web server for XHM. In the final section we report some example results using our system on three cDNA probe sets for human, mouse and rat, and one oligonucleotide probe set for mouse. Implementation The XHM system queries the given nucleotide sequences (for example, a list of differentially regulated genes from DNA microarray experiments) against a database representing all possible targets (transcripts) in the organism being studied, and produces a list of all targets that can hybridize to each input sequence under the defined criteria. In this section we evaluate databases for use in the XHM system and we evaluate whether BLAST can be used to identify potential cross-hybridizing genes. Definitions For the calculation of melting temperature, the following formula is used [ 15 ]: Tm = 81.5 + 16.6 * log [ Na + ] + 41 * ( numG + numC )/ n - 500/ n where [ Na + ] is the Na + concentration, numG and numC is the number of Gs and Cs in the given alignment, and n is the total number of nucleotides aligned. We differentiate between two kinds of sequence similarity cutoffs for cross-hybridization, based on the situations described for oligonucleotide microarrays [ 3 ]: • Type A similarities for sequence segments of a certain minimum length with a defined minimum percentage identity (long alignments with mismatch). • Type B similarities for identical segments of some minimum length (short perfect match). To perform the searches in the different databases, NCBI BLAST version 2.2.1 (Mon Jul 9 14:02:00 EDT 2001) [ 16 ] was used. The default settings of blastn are used unless specified otherwise. This includes using the DUST filter for masking lowcomplexity regions. Databases and sequences In this study we used three cDNA clone sets (corresponding to three cDNA sets spotted on microarrays). The 40 k Homo sapiens (Hs) and the 14 k Rattus norvegicus (Rn) clone sets are I.M.A.G.E. Consortium [LLNL] cDNA clones [ 17 , 18 ] obtained from Research Genetics (Huntsville, AL). The 15 k Mus musculus (Mm) clone set is from NIA [ 19 ]. In addition, oligonucleotide sequences (Mouse Genome Array Ready Oligonucleotide Set) from QIAGEN Operon [ 20 ] have been used to test the application. The details of the databases used to search for possible cross-hybridizations are as follows: • RefSeq Version 2 – RefSeq mRNA collection [ 21 ]. Number of sequences: 25377/21200/21724 (mouse/rat/human). • UniGene – UniGene Unique, build 129/123/164 (mouse/rat/human) [ 22 ]. Number of sequences: 87495/50137/118326 (mouse/rat/human). • TIGR gene index [ 23 ] – Only tentative consensus sequences (TCs), version 020403/042503/020503 (mouse/rat/human). Number of sequences 58129/51330/187287 (mouse/rat/human). • BeGIn – Bergen Gene Index version 1.0. Number of sequences: 100654/49285 (mouse/rat). BeGIn is an alternative database we have developed (using tools described in [ 24 ]), by producing consensus sequences from each cluster in UniGene (build 129/123 for mouse/rat). In order to achieve high specificity and sensitivity, we favored the databases where the highest number of probes were represented (completeness) and multiple matches per probe were minimized (minimum redundancy). Our criteria for choosing an appropriate database for a given organism was based on the results obtained for our probe sets. The presence of alternative splice forms in either the probe or the target set may complicate analysis. We do not consider this explicitly in the current study. Overview of the XHM system The basis of the XHM system is a BLAST search (see Figure 1 ). The BLAST search provides one or several alignments between the probe sequence and each of the possible cross-hybridizing target sequences. The alignments are analyzed with respect to whether they fulfill the user-defined criteria for type A or type B matches. The XHM system presents to the user a list of matches satisfying the criteria, and in addition information on GC-content in similar regions, estimated melting temperature (Tm) and position on the sequence (proximity to 3' or 5' end). Although the estimated Tm may not be completely accurate under microarray hybridization conditions, particularly on solid surfaces, it can give a relative measure of the impact of the potential cross-hybridization detected. An expert user may use the information to assess the likelihood and importance of the cross-hybridization effect. Appropriateness of BLAST Experiments were performed to assess whether BLAST is able to identify potentially cross-hybridizing targets. The analysis was done by generating simulated targets ( T 1 ... T n ) for a number of probes ( P 1 ... P n ). The simulated targets were ensured to satisfy the criteria described in [ 3 ] (50 bp sequences with at least 75% identity or 15 bp identical sequence). BLAST was then used to query the original probes against a large database containing the designed targets. It was then checked whether or not T i was contained in P i 's hitlist. We found that type A matches can be missed if they do not contain any one stretch identical to the probe of length larger than BLAST's initial word length. Our experiments also showed that, depending on the probe type and the criteria used, we sometimes had to allow BLAST to generate very long lists of hits and using very high (permissive) cutoffs on E-value in order to cover the intended targets. Part of the reason for this is that BLAST is designed to identify homologous sequences and the sequences satisfying the cross-hybridization criteria need not necessarily receive significant scores or E-values. Our conclusion was that BLAST can be used, but to achieve optimal results, non-default parameters for initial word size and mismatch penalty should be applied. This was taken into consideration in the web-server. A web server A web interface has been designed with two entry points for accessing cross-hybridization information (see Figure 1 ). The user may either query a database of precomputed alignments, or compute new alignments using a real-time BLAST search. Both versions have parameters for the different thresholds and output alternatives. For ease of use, it is possible to run queries with no parameters explicitly specified. When querying with a large number of clones against a large database, running BLAST may be time consuming. Therefore, we pre-run BLAST with different clone-sets against different databases, and store the results. In this way the user may experiment with different thresholds for a set of clones repeatedly, and get the results within seconds. Nucleotide sequences or GenBank accession numbers may be used as input to the system when running the real-time BLAST searches. Searching using the precomputed BLAST alignments does not accept nucleotide sequences as input. User-adjustable input parameters Some of the parameters are shared among the two main versions of the XHM system. These include minimum length and minimum percentage similarity for type A hits (defaults are 75% over 50 bp) and minimum length for type B hits (default is 15 bp), based on the findings described in [ 3 ], Na + concentration (default is 0.1 M), and size of GC-clamp (default is 10). The Na + concentration is used to calculate melting temperature, and the size of the GC-clamp is used to plot the GC-content throughout the query sequence. In addition, the real-time BLAST version also contains BLAST specific user-adjustable parameters. Main parameters include threshold on E-value (default is 10), number of alignments in the output from BLAST and whether DUST (low complexity) filter should be used. The user can adjust other BLAST specific parameters including gap opening and extension penalties, initial word size, mismatch penalty and whether or not to allow gapped alignments. Output The output from the XHM system consists of different parts (see Figure 2 ). If there are several input sequences (batch query), the results are given for each sequence individually. For each probe the system generates a plot of GC-content along the sequence. This GC-plot may help the user to identify areas in the sequence with high GC content in order to evaluate the importance of the potential cross-hybridization detected. For each hit (possible hybridization), XHM presents the name and identifier of the hit, identity tuples (from BLAST – number of identical bases and number of bases in total in the BLAST alignment, or in a sub-alignment), start and end position on the probe, calculated Tm, percentage GC in alignment and type of hit (A or B). Results Choice of database Experiments using the three cDNA sets and the oligonucleotide set as input to the XHM system yielded the results shown in Table 1 . Using a 70% identity threshold for the type A similarities, in the rat clone set the Rat Gene Index from TIGR appears to be the best choice. For the mouse clone set it appears that the BeGIn database is the best choice, and for the human clone set it seems that RefSeq or UniGene Unique may be two good choices. These evaluations were based on completeness (high number of probes represented) and minimum redundancy of the database. We used only the tentative consensuses (TCs) of the Gene Indices databases from TIGR. Using the "full" versions decreased the number of probes having zero hits, but substantially increased the number of probes having two or more hits (results not shown). Application to microarray probes As a practical experiment, we tested the XHM system on the cDNA probe sets for human, mouse and rat, and the mouse oligonucleotide set from QIAGEN. For the cDNA sets we used the following thresholds: 70% over 200 nucleotides for type A similarities, and 25 nucleotides for the type B similarities. For the oligonucleotides we used 70% identity over the whole oligonucleotide sequence ( type A ) and 20 nucleotides perfect identity ( type B ). Choosing the parameters is not trivial, and these thresholds, which are in the lower range of % similarities leading to cross-hybridizations, are meant as examples of a possible configuration. The XHM tool allows for full flexibility to experiment with the settings. The reason why we chose to consider a 200 nucleotides stretch for the cDNA probes is that sometimes these sequences contain errors, especially toward the ends. Results are shown in Table 1 . Using RefSeq, we observed that even though a substantial number of the 14 k rat cDNA probes (63.3%) had no type A hit, 809 (about 16.3% of the probes actually represented in RefSeq) had two or more hits, indicating potential cross-hybridization. In most cases, a single hit represents the probe sequence itself, and does not represent a potential cross-hybridization. Looking at a more complete database, the TIGR Rat Gene Index, only 700 (5.2%) probes had no hits, and 2712 (21.3% of the probes represented in the database) had two or more hits. A conclusion from this was that the number of rat probes having potential cross-hybridizing partners was between 16% and 21%. For the mouse cDNA probes the cross-hybridization numbers appeared to be higher. Using RefSeq, the number of probes having two or more type A hits was 3101 or 34.2% of the probes represented in the database. RefSeq is relatively incomplete, so this number may be a conservative estimate on the cross-hybridization occurrence. For the BeGIn database only 725 probes had no hits (5.3%) and 5211 probes (about 40% of the probes in the database) had two or more hits. Even though as many as 47.3% of the mouse probes were not found in the TIGR Gene Index, the proportion of probes found in the database that had two or more hits was 59%. This number is most likely an artifact caused by redundancy in the database. The difference in number of possible hybridizations found in mouse versus rat is most likely due to the fact that more mouse sequences are available. The human cDNA probes seems to have a lower cross-hybridization potential than the mouse probes. In UniGene Unique and RefSeq, looking only at the human probes that were found in the databases, just above 30% of them had two or more hits. The results using TIGR Human Gene Index suggested that almost 70% of the probes found in the database could cross-hybridize, but this is most likely an artifact caused by redundancy. The mouse oligonucleotide set is clearly less prone to cross-hybridization. Using the UniGene Unique database, we observed that 14% of the probes found in the database had two or more hits, whereas the corresponding number for the 15 k mouse cDNA probe set using UniGene Unique was 45%. Discussion A flexible tool for assessing the cross-hybridization potential of microarray probes has been developed and made available. Several transcriptome databases can be used for searching, and more may be added upon request. Using the XHM tool, analysis of three cDNA microarray probe sets and one oligonucleotide probe set revealed that a high proportion of the cDNA probes can potentially cross-hybridize with one or more other transcripts in the organism. As expected, compared to the cDNA probes, a smaller percentage of the mouse oligonucleotide probes showed a potential for cross-hybridization. This is because the oligonucleotide probe sequences are much shorter (69-mers), designed from specific regions of cDNAs to minimize cross-hybridizations. Despite increasing use of the more specific oligonucleotide arrays, cDNA sequences including full-length clone sets [ 25 , 26 ] are widely used in production of microarrays. Depending on the completeness and redundancy levels of the transcriptome databases used, with the chosen cutoff for type A similarities (at least 70% identity), 15–45% of the cDNA probes showed hybridization with two or more apparently different transcripts (disregarding the results using the TIGR Gene Indices). This high percentage of potentially cross-hybridizing genes suggests that it is essential to carefully validate results from microarray experiments, particularly where cDNA clones are used to prepare arrays. Although cross-hybridization is known as one of the main sources of errors of cDNA microarrays, the high proportions of cross-hybridizing genes detected in our test are likely to be overstated by the possible redundancies in the databases (not all entries represent unique genes). Also one may argue that the 70% identity threshold is somewhat low. Whether two different genes that are candidates for cross-hybridization actually lead to erroneous results in hybridization experiments will depend on factors such as the level of sequence identity, the stringency of the hybridization and the relative abundances of the transcripts. For example, two potentially cross-hybridizing genes do not necessarily pose a problem unless both are expressed in the tissue or cell-line analyzed. Quick inspection of the hit-list produced by the XHM tool for a typical input of differentially regulated genes will help in identifying significant noise from cross-hybridizations. A main advantage of the XHM tool is that it allows the user to perform searches at various stringencies to detect potential cross-hybridizations. Candidate genes from microarray analysis that show potential cross-hybridizations using the database search may then be further checked using other methods such as RT-PCR and Northern blot. Conclusions We have shown that a significant proportion of probes used in cDNA microarray analysis may show cross-hybridization with non-target sequences. We have developed a flexible tool, XHM, suitable for detecting potential cross-hybridization artifacts during microarray data analysis. The tool may also be used to select specific probes for preparation of microarrays. Availability The XHM system is freely available at . Program code for academic use can be supplied upon request to the author. Author's contributions KF implemented the system and made a draft of the manuscript. FY and AL contributed with ideas and proofread the manuscript. IJ has formulated and supervised the work, and edited the manuscript. All authors have read and approved the final manuscript. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517492.xml |
385220 | Single Locus Controls Majority of Armor Evolution in Two Populations of Sticklebacks | null | The astounding diversity of life—different body shapes and sizes, physiologies, and behaviors—stems from the accumulation of genetic changes through the process we call evolution. But catching a glimpse into the process of evolution at the gene level is difficult, mostly because significant changes to the plant and animal species of today happened a long time ago. Nevertheless, biologists are keen to understand exactly how evolution progresses. For example, how many genes must be altered before noticeable shifts in appearance can be seen? Is evolution the result of changes in many genes with small additive effects, or of just a few mutations that exert a strong influence? Complete and low armor stickleback morphs, Friant, California To tackle these questions, Pamela Colosimo and colleagues turned to threespine stickleback fish, a longtime favorite model system of evolutionary biologists because of its relatively youthful evolutionary history. At the end of the last ice age 10,000 years ago, when glaciers all over the Northern Hemisphere began to melt, small populations of these originally marine-dwelling fish became trapped in newly formed lakes. There, isolated stickleback colonies adapted to new ecological conditions—different predators, food availability, water chemistry, and temperature—and now look distinctly different from their marine ancestors. One of the most obvious changes in appearance is in their body armor—they come in three distinct types, or “morphs.” Marine sticklebacks are covered from head to tail with rows of tightly packed boney plates (a complete morph), while those found in freshwater lakes have fewer body plates (a partial morph) or almost none at all (a low morph). Colosimo and colleagues found that a single region of the genome is largely responsible for the dramatic changes in plate morph, and that this is true for two widely separated populations of independently evolving freshwater sticklebacks. To uncover the genomic regions that affect armor, Colosimo's team crossed fully armored marine sticklebacks from Japan with deep-water, or benthic, low morph fish from Paxton Lake in British Columbia, Canada. They then “mapped” the full genome of second generation offspring using 160 known genetic markers, or loci, as guideposts for distinct regions of the genome—loci that are inherited along with differences in the overall type of plating, and individual plate number and size. The team found that one such locus explained 75% of the variation in plate morphs. Offspring that carried two alleles—versions of the gene—from their marine grandparents, genotype AA, were almost always fully plated. Those that inherited two copies of the allele from their benthic progenitors, aa, were mostly low morphs with very little plating. And Aa heterozygous fish (with one allele from each population) had mostly full or partial plates. Colosimo and colleagues also found three other regions in the genome that significantly affected the number and size of plates. These modifiers had an additive effect—the more benthic alleles inherited, the fewer and smaller the plates; more marine alleles caused a trend toward greater armor. But is this genetic architecture the same for every independently evolving population of lake-bound sticklebacks in North America? Or did the geographically isolated freshwater groups loose their plates through mutations in different genes? Colosimo and colleagues mapped the genome of a population of sticklebacks from Friant, California, which is 800 miles away from Paxton Lake, and found that the same major locus seemed to be controlling plate morph there as well. Crossing a low morph from Friant with a low morph from Paxton yielded only offspring with very little armor. Further, some of the modifiers uncovered in the Paxton fish were also acting on the Friant sticklebacks. So, though these two populations of fish have been separated for 10,000 years, loss of armor in both groups probably stemmed from changes in the same genetic pathway. Without knowing the precise sequence of these genes, it is impossible to tell exactly how and when the alleles that reduce armor arose. Small numbers of individuals with genes causing less plating could have been present in ancestral populations of marine sticklebacks when they were originally locked in newly formed lakes. Alternatively, reduced armor could have arisen independently in different lakes following isolation if, for example, some genes that control armor are predisposed to mutation, or certain armor-related mutations are more advantageous than others. But however it happened, this study clearly shows that dramatic morphological evolution can result from a small number of genetic changes. Further study of this classic system should provide a detailed picture of the genes involved, and of the molecular events that underlie morphological changes in natural populations evolving in new environments. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC385220.xml |
546193 | Stainable hepatic iron in 341 African American adults at coroner/medical examiner autopsy | Background Results of previous autopsy studies indicate that increased hepatic iron stores or hepatic iron overload is common in African Americans dying in hospitals, but there are no reports of hepatic iron content in other cohorts of African Americans. Methods We investigated the prevalence of heavy liver iron deposition in African American adults. Using established histochemical criteria, we graded Perls' acid ferrocyanide-reactive iron in the hepatocytes and Kupffer cells of 341 consecutive African American adults who were autopsied in the coroner/medical examiner office. Heavy staining was defined as grade 3 or 4 hepatocyte iron or grade 3 Kupffer cell iron. Results There were 254 men and 85 women (mean age ± 1 SD: 44 ± 13 y vs. 48 ± 14 y, respectively; p = 0.0255); gender was unstated or unknown in two subjects. Approximately one-third of subjects died of natural causes. Heavy staining was observed in 10.2% of men and 4.7% of women. 23 subjects had heavy hepatocyte staining only, six had heavy Kupffer cell staining only, and one had a mixed pattern of heavy staining. 15 subjects had histories of chronic alcoholism; three had heavy staining confined to hepatocytes. We analyzed the relationships of three continuous variables (age at death in years, hepatocyte iron grade, Kupffer cell iron grade) and two categorical variables (sex, cause of death (natural and non-natural causes)) in all 341 subjects using a correlation matrix with Bonferroni correction. This revealed two positive correlations: hepatocyte with Kupffer cell iron grades (p < 0.01), and male sex with hepatocyte iron grade (p < 0.05). We also analyzed the relationship of steatosis, inflammation, and fibrosis/cirrhosis in 30 subjects with heavy iron staining using a correlation matrix with Bonferroni correction. There were significant positive correlations of steatosis with inflammation (r = 0.5641; p < 0.01), and of inflammation with fibrosis/cirrhosis (r = 0.6124; p < 0.01). Conclusions The present results confirm and extend previous observations that heavy liver iron staining is relatively common in African Americans. The pertinence of these observations to genetic and acquired causes of iron overload in African Americans is discussed. | Background Hepatic iron overload was detected by Perls' acid ferrocyanide staining and atomic absorption spectrometry at autopsy in more than one percent of African American adults who died in hospitals [ 1 , 2 ]. In one study [ 1 ], liver specimens from 326 unselected adult African Americans subjects were stained for iron; liver iron was quantified using atomic absorption spectrometry in subjects in whom increased stainable iron was observed. Four subjects (1.2%), two men and two women aged 50 to 63 years, had hepatic iron indices adjusted for previous erythrocyte transfusion that were ≥ 1.9 (range 1.9 – 5.6) [ 1 ]. In a second study [ 2 ], hepatic iron concentrations of liver tissue from autopsies in 99 African Americans were quantified. Thirty-one (31.3%) had an elevated hepatic iron concentration, including nine (9.1%) who had an hepatic iron concentration greater than twice the upper limit of normal and no evident cause of secondary iron overload [ 2 ]. These results suggest that iron overload not attributable to erythrocyte transfusion is relatively common in African Americans. In contrast, screening programs that included cohorts of African Americans presumably representative of the general African American population identified a much lower proportion of subjects with possible iron overload [ 3 - 5 ] than is suggested by the results of hospital autopsy series [ 1 , 2 ]. These studies used an elevated transferrin saturation phenotype criterion generally regarded as the best for screening whites for HFE -associated hemochromatosis [ 3 - 5 ]. In these studies, ≤ 0.9% of African Americans adults had a positive screening result(s), and ≤ 0.09% were subsequently demonstrated to have hemochromatosis or iron overload [ 3 - 5 ]. Taken together, these observations suggest that previous reports of increased hepatic iron content in African Americans dying in hospital may have overestimated the prevalence of non-transfusion iron overload in African American adults in the general population, or that the ideal phenotype for screening African Americans for primary iron overload differs from that which is optimal for screening whites for HFE -associated hemochromatosis. Thus, we graded Perls' acid ferrocyanide-reactive iron in the livers of 341 consecutive African American adults who underwent autopsy in the coroner/medical examiner office. We selected this population for study because such subjects are more representative of the general African American population than persons who died in hospital. We then compared these observations with autopsy results reported previously in African Americans who died in hospitals [ 1 , 2 ]. The pertinence of these observations to genetic and acquired causes of iron overload in African Americans is discussed. Methods Selection of study subjects The performance of this study was approved by the Institutional Review Board of the University of Alabama at Birmingham and by the Coroner/Medical Examiner's Office of Jefferson County, Alabama. We evaluated formalin-fixed tissue obtained in 361 consecutive, unselected coroner/medical examiner autopsy cases of African-American adults (age >18 years) in Jefferson County, Alabama; deaths in all of the present cases occurred during the interval 1998 – 2002. Each subject was identified as African American by his/her previous medical histories or legal records, or by the coroner/medical examiner staff. Most autopsies were performed on the same day that the respective bodies were available for evaluation at the Coroner/Medical Examiner's Office; other autopsies were completed within ~ 18 hours. All tissues were placed directly in fixative at the time of collection at autopsy. Liver was not available or was not interpretable due to autolysis in 20 cases. Available records in each case were reviewed; age at death, sex, summary of known illnesses, and cause of death were tabulated for all cases for which liver tissue was available. Histologic technique, iron grading, and liver morphology Tissues obtained at autopsy were routinely fixed in 10% neutral buffered formalin. Triplicate sections of paraffin-embedded liver were prepared. One section was stained with hematoxylin and eosin, another with Perls' acid ferrocyanide technique to demonstrate non-heme ferric iron [ 6 ], and a third with Masson trichrome technique. Appropriate positive and negative control specimens were prepared with each staining batch and reviewed. All slides were simultaneously reviewed by three investigators, and the iron grades assigned in each case represent their consensus opinions. Hepatocellular iron was graded according to these criteria: grade 0 – no visible iron; grade 1 – iron visible in very few hepatocytes; grade 2 – iron visible in 5 – 10% of hepatocytes; grade 3 – iron visible in ≥ 40% of hepatocytes; and grade 4 – abundant iron visible in most hepatocytes [ 7 ]. Kupffer cell iron was graded according to these criteria: grade 0 – no visible iron in Kupffer cells; grade 1 – iron visible in ≤ one-third of Kupffer cells; grade 2 – iron visible in one third to ≤ two-thirds of Kupffer cells; and grade 3 – abundant iron visible in more than two-thirds of Kupffer cells [ 7 ]. Hepatocyte or Kupffer cell iron of grades 0 or 1 was defined as normal. Increased stainable iron was defined as hepatocyte and/or Kupffer cell iron grade ≥ 2 [ 7 ]. Heavy iron staining was defined as hepatocyte iron grade of 3 or 4, or Kupffer cell iron grade of 3. Steatosis, inflammation, and fibrosis/cirrhosis were assessed as described in detail elsewhere [ 8 ]; these abnormalities were graded as absent (0) or present (+). We designated a gradient of stainable iron in hepatocytes from the periportal area decreasing towards the hepatic venule as present or absent; visualization of a gradient required hepatocyte iron staining of grade ≥ 2 [ 9 , 10 ]. The presence or absence of hepatic cirrhosis was determined using Masson trichrome-stained specimens as described previously [ 8 ]. Statistical considerations The present data set consisted of observations in 341 subjects and their respective livers. Analyses were performed with a computer spreadsheet (Excel 2000 ® , Microsoft Corp., Redmond, WA), and a statistical program (GB-Stat ® v. 10.0, 2003, Dynamic Microsystems, Inc., Silver Spring, MD). Descriptive data are displayed as enumerations, percentages, mean ± 1 S.D., medians, and ranges. Frequency values were compared using chi-square analysis or Fisher exact test, as appropriate. Mean values were compared using student t-test. Some data were analyzed using a correlation matrix with Bonferroni correction. Values of p < 0.05 were defined as significant. Results General characteristics of study subjects There were 254 men (mean age 44 ± 13 y; median age 42 y; range 26 – 99 y), and 85 women (mean age 48 ± 14 y; median age 45 y; range 26 – 89 y). The mean age of men was lower than that of women (p = 0.0255). There were two subjects for whom gender was unstated or unknown. Approximately one-third of subjects died of natural causes (Table 1 ). Table 1 Causes of death of 341 African Americans autopsied in the coroner/medical examiner office. 1 Causes of Death Subjects, n Percentage Homicide 130 38.1 Natural Cause 107 31.4 Accident 90 26.4 Unknown 7 2.1 Suicide 5 1.5 Not Stated 2 0.6 1 Chronic alcoholism was listed for 15 subjects (the respective cause of death in each subject was ruled to be "natural cause"). Two subjects were reported to have diabetes mellitus; one had grade 3 hepatocyte and grade 1 Kupffer cell iron. No subject was reported to have iron overload, hemochromatosis, heritable or acquired forms of anemia, or treatment with erythrocyte transfusion. Liver iron grades In the 341 evaluable subjects, there was a significant positive correlation of grades of stainable iron in hepatocytes and Kupffer cells across the 341 evaluable subjects (Pearson r coefficient = 0.2370; p < 0.0001). However, the mean hepatocyte iron grade was 0.83 ± 0.96; the mean Kupffer cell iron grade was 0.32 ± 0.32. Increased hepatocyte iron grades were observed in 64 men (25.2%) and 8 women (9.4%) (p = 0.0021). Increased Kupffer cell iron grades were observed in 20 men (7.9%) and one woman (1.2%) (p = 0.0266) (Tables 2 , 3 ). Table 2 Iron grades of 341 Perls' acid ferrocyanide-stained liver sections. 1 Grade No. of subjects with hepatocyte staining (%) No. of subjects with Kupffer cell staining (%) 0 164 (48.1) 270 (79.2) 1 104 (30.5) 50 (14.7) 2 49 (14.4) 14 (4.1) 3 21 (6.2) 7 (2.1) 4 3 (0.9) not available 1 1 The present grading system does not include Kupffer cell iron staining greater than grade 3. In all subjects, the mean hepatocyte grade (± 1 SD) was 0.83 ± 0.96; the mean Kupffer cell grade was 0.32 ± 0.31. A gradient of stainable iron in hepatocytes from the periportal area decreasing towards the hepatic venule was observed in 56 subjects. Table 3 Histologic findings in 30 African American subjects with heavy liver iron staining 1 Age, years Sex Hepatocyte iron grade Kupffer cell iron grade Steatosis Inflammation Fibrosis/cirrhosis 26 M 3 1 0 0 0 27 M 1 3 0 0 0 28 M 3 0 0 0 0 29 M 3 0 0 0 0 30 M 3 1 0 0 0 34 M 3 0 0 0 0 34 M 0 3 0 0 0 37 M 3 2 + + 0 37 M 3 1 + + 0 39 M 3 1 0 0 0 39 M 3 1 0 + + 40 M 2 3 + + 0 42 M 3 0 0 0 0 43 M 3 0 0 0 0 44 M 4 1 0 + 0 44 M 4 0 0 + + 2 46 M 3 0 0 + 0 49 M 3 0 0 0 0 50 M 1 3 0 + 0 52 M 3 0 + + + 55 M 3 0 0 0 0 59 M 3 0 + + 0 59 M 0 3 0 + + 63 M 3 1 + + + 67 M 2 3 0 + + 91 M 3 0 + + + 2 33 F 3 3 0 0 0 50 F 4 0 0 + + 2 51 F 3 0 + + + 54 F 3 0 0 0 0 1 Heavy iron staining was defined as hepatocyte iron grade of 3 or 4, or Kupffer cell iron grade of 3. Steatosis, inflammation, and fibrosis/cirrhosis were assessed as described in detail elsewhere [8]; these abnormalities were graded as absent (0) or present (+). 2 These subjects had hepatic cirrhosis [8]. Heavy iron staining was observed in 30 subjects (8.8%), including 10.2% of men and 4.7% of women (p = 0.1202). The mean age of men and women with heavy iron staining was similar: 45 ± 15 y and 47 ± 9 y (p = 0.7389). 24 subjects (7.1%) had grade 3 or 4 hepatocyte iron; of these, one also had grade 3 Kupffer cell iron. Seven subjects (2.1%) had grade 3 Kupffer cell iron; of these, one also had grade 3 hepatocyte iron. Altogether, 23 subjects had heavy iron staining in hepatocytes only (Fig. 1 ), six subjects had heavy iron staining in Kupffer cells only (Fig. 2 ), and one subject had a mixed pattern of heavy hepatocyte and Kupffer cell iron staining (Fig. 3 ). The causes of death of the 30 subjects who had heavy iron staining were similar to those of subjects with lower iron grades (data not shown). Figure 1 Photomicrograph of non-cirrhotic liver stained with Perls' technique. Liver of a 44 year-old African American man who died of pneumonia. There is a predominance of iron staining (grade 4) in hepatocytes. Original magnification 40×. Figure 2 Photomicrograph of non-cirrhotic liver stained with Perls' technique. Liver of a 34 year-old African American man who died of homicide. There is a predominance of iron staining (grade 3) in Kupffer cells; there is faint diffuse staining of hepatocytes (grade 1). Original magnification 40×. Figure 3 Photomicrograph of non-cirrhotic liver stained with Perls' technique. Liver of a 33 year-old African American woman who died of accidental trauma. There is heavy iron staining in hepatocytes (grade 3) and Kupffer cells (grade 3). Original magnification 40×. We analyzed the relationships of three continuous variables (age at death in years, hepatocyte iron grade, and Kupffer cell iron grade) and two categorical variables (sex, cause of death (natural and non-natural causes)) using a correlation matrix with Bonferroni correction. This revealed two significant positive correlations: hepatocyte iron grade with Kupffer cell iron grade (p < 0.01), and male sex with hepatocyte iron grade (p < 0.05). Histologic findings in 30 subjects with heavy iron staining These subjects were comprised of 26 men and 4 women (Table 3 ). Hepatic steatosis was observed in eight subjects (26.7%), hepatic inflammation was observed in 16 subjects (53.3%), and hepatic fibrosis or cirrhosis was observed in nine subjects (30.0%). However, 14 subjects (46.7%) did not have steatosis or inflammation; none of these 14 subjects had fibrosis/cirrhosis. We analyzed the relationship of hepatocyte and Kupffer cell iron grades, steatosis, inflammation, and fibrosis/cirrhosis in these 30 subjects (Table 3 ) using a correlation matrix with Bonferroni correction. There was a significant negative correlation of hepatocyte and Kupffer cell iron grades (r = -0.7368; p < 0.01). There was a significant positive correlation of steatosis with inflammation (r = 0.5641; p < 0.01). There was a significant positive correlation of inflammation with fibrosis/cirrhosis (r = 0.6124; p < 0.01). Subjects with chronic alcoholism Fifteen subjects had histories of chronic alcoholism; three of these had heavy liver iron staining. In each case, iron staining was confined to hepatocytes. The percentage of subjects with heavy iron staining was similar in 15 subjects reported to have chronic alcoholism and in 326 subjects not reported to have chronic alcoholism (20.0% vs. 8.3%; p = 0.1356). Three persons who had histories of chronic alcoholism also had hepatic cirrhosis. A 36 year-old man had grade 0 hepatocyte and grade 0 Kupffer cell iron. A 46 year-old man had grade 2 hepatocyte iron and grade 0 Kupffer cell iron. A 50 year-old woman had grade 4 hepatocyte iron and grade 0 Kupffer cell iron (Fig. 4 ). Figure 4 Photomicrograph of cirrhotic liver stained with Perls' technique. Liver of a 50 year-old African American woman with a history of chronic alcoholism. There is a predominance of iron staining (grade 4) in hepatocytes, and prominent staining of bile ductule cells. Micronodular cirrhosis and moderate-severe steatosis were also present. Original magnification 100×. Subjects with cirrhosis Five subjects had hepatic cirrhosis (1.5%). Three had histories of chronic alcoholism (described above). One of these three subjects, a 50 year-old woman, had grade 4 hepatocyte iron and grade 0 Kupffer cell iron (Fig. 4 ). Two other subjects, neither of whom had a history of alcoholism, also had hepatic cirrhosis. One was a 44 year-old man with grade 4 hepatocyte iron who died of pneumonia (Fig. 1 ; Table 3 ). The other was a 91 year-old man with grade 3 hepatocyte iron who died of intracranial hemorrhage (Table 3 ). Discussion Heavy hepatocyte or Kupffer cell iron staining was observed in 8.8% of the present subjects. This is consistent with prevalence estimates of hepatic iron overload reported in hospital autopsy series of African Americans from other geographic areas [ 1 , 2 ]. The present subjects were relatively young, on the average, and approximately two-thirds died of non-natural causes. In contrast, the subjects in previous hospital autopsy series were much older, on the average, and most died of natural causes [ 1 , 2 ]. In one hospital autopsy series, all subjects but one were men [ 2 ]. Although there was a predominance of men in the present study, our series included 85 women. We observed that the percentages of men with increased hepatocyte or Kupffer cell iron grades were greater than those of women. This is in agreement with the greater mean iron stores of African American men than women detected in assessments of iron nutrition, and with the predominance of men in clinical and autopsy series of African Americans with primary iron overload [ 1 , 7 , 11 , 12 ]. Altogether, the present subjects may be more representative of African American adults in the general population than those in hospital autopsy series [ 1 , 2 ], although there may be fewer available observations regarding medical history in the present cases than in African Americans who died in hospital [ 1 , 2 ]. Microscopic estimation of liver iron content correlates well with atomic absorption spectrometry measurements in subjects in whom the histologic distribution of hepatic iron and clinical circumstances suggest hemochromatosis, i.e. , predominance of hepatocyte iron and no apparent explanation for iron overload [ 9 , 13 - 15 ]. These histologic criteria were pertinent to 77% of the present subjects. On the other hand, the relationship of iron grades and quantitative liver iron measurements is not well documented in subjects in whom hepatic iron deposition occurs predominantly in macrophages, like 20% of the present subjects. Hepatic iron concentrations and indices have been used as conservative, surrogate diagnostic criteria for primary iron overload in African Americans [ 1 , 2 , 7 , 16 ], although there has been no validation of their use in such cases. Further, some African Americans who had iron overload demonstrated by therapeutic phlebotomy had normal hepatic iron concentrations and indices [ 7 , 17 ]. Elevated hepatic iron indices have also been reported to occur in a variety of other conditions [ 18 - 20 ]. Primary iron overload in African Americans is often associated with preferential deposition of iron in macrophages in multiple organs [ 1 , 7 , 21 ]. In some cases, this is associated with the inheritance of the Q248H missense mutation of the ferroportin gene FPN1 [ 17 , 21 , 22 ]. Two of thirteen (15.4%) African American iron overload index patients and two of 39 (5.1%) African American control subjects who reside in central Alabama were heterozygous for FPN1 Q248H [ 21 ]. Nine of the present 341 (6.0%) subjects had heavy iron staining confined to Kupffer cells. Thus, FPN1 Q248H could account for heavy iron staining in some of the present subjects. Other African Americans with primary iron overload have a predominance of hepatocyte iron deposition. This is consistent with hemochromatosis phenotypes associated with HFE genotypes typical of hemochromatosis in whites ( HFE C282Y/C282Y or C282Y/H63D) [ 16 , 21 ], or with common types of hemoglobinopathy or thalassemia [ 7 , 21 ]. Other African Americans with primary iron overload and a predominance of hepatocyte iron staining have missense mutations of the hemojuvelin gene HJV on Ch1q [ 23 ] or the erythroid-specific 5-aminolevulinate synthase gene ALAS2 on ChX [ 24 , 25 ]. Other putative African iron overload alleles may account for iron overload in some cases [ 26 ]. However, performing DNA analyses to detect mutations of iron-associated genes was beyond the scope of the present work. Acquired disorders account for increased hepatic iron deposition in some African Americans. In the present study, we did not observe any subject who had heavy liver iron staining and fibrosis or cirrhosis who did not also have hepatic steatosis or inflammation. Chronic viral hepatitis C occurs in approximately 1.8% of the overall U.S. population [ 27 ], and the prevalence of chronic hepatitis C is significantly greater in African Americans than whites in the U.S. [ 27 , 28 ]. A greater proportion of African Americans than persons of other races respond to chronic hepatitis C infection with an increase in iron stores, after adjustment for age, alcohol intake, gender, menopausal status, education, body mass index, and poverty index [ 29 ]. More than half of the present subjects who had heavy iron staining had hepatic inflammation. It is plausible that some of these had viral hepatitis C, although this is unproven. More than one-quarter of the present subjects who had heavy iron staining also had hepatic steatosis. The prevalence of non-alcoholic steatosis and steatohepatitis is lower in African Americans than in whites [ 30 , 31 ], although some risk factors for non-alcoholic hepatic steatosis and steatohepatitis (obesity, insulin resistance, and diabetes mellitus) are significantly greater in African Americans than in whites in the U.S. [ 30 - 32 ]. Taken together, these findings suggest the development of hepatic fibrosis or cirrhosis in African Americans who have heavy hepatic iron deposition may require the synergistic effects of hepatic steatosis or inflammation. Iron overload sometimes develops spontaneously or after repeated erythrocyte transfusion in African Americans with heritable or acquired anemia, or with myelodysplasia or acute leukemia [ 7 , 15 , 33 - 41 ]. Although there were no reports of heritable or acquired anemia or of multiple erythrocyte transfusions in the present subjects, such circumstances were frequent in hospital autopsy series of African Americans [ 1 , 2 ]. Three of the five present subjects with micronodular cirrhosis had a history of chronic alcoholism. African Americans are at greater risk than whites for developing several alcohol-related conditions, including hepatic cirrhosis [ 42 , 43 ]. In the present study, however, the prevalence of heavy liver iron staining was similar in subjects with and without histories of chronic alcoholism. Conclusions We conclude that heavy liver iron staining is common in African American adults who were autopsied in the coroner/medical examiner office. The different histologic patterns of heavy liver iron staining we observed in the present subjects are consistent with the phenotypic and genotypic heterogeneity of primary iron overload in African Americans [ 21 ] and with the phenotypic heterogeneity of iron overload of other causes [ 7 , 29 , 33 - 41 ]. However, the present results do not demonstrate specific genetic or acquired causes for heavy liver iron staining in individual subjects. Further, the present results do not prove that the present subjects with heavy liver iron staining had systemic iron overload. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JCB conceived and designed the study, reviewed and graded the liver specimens, contributed to the statistical analyses of data, and contributed to writing the manuscript. RTA reviewed and graded the liver specimens, contributed to the statistical analyses of data, and contributed to writing the manuscript. AKR reviewed and graded the liver specimens and contributed to writing the manuscript. RMB provided information on the autopsy cases, provided the liver specimens, reviewed the histology of selected liver specimens, and contributed to writing the manuscript. All authors approved of the manuscript in its final form. Pre-publication history The pre-publication history for this paper can be accessed here: | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546193.xml |
523858 | Tuberculous peritonitis in a case receiving continuous ambulatory peritoneal dialysis(CAPD) treatment | Background Tuberculosis continues to be an important health problem in the world. Besides pulmonary involvement extrapulmonary involvement becomes an affair in developing countries, even in developed countries. Case presentation A thirty-six year old male patient was admitted with abdominal pain, diarrhea, nausea, vomiting and fever which had started one week before. The patient had been followed up with predialisis Chronic Renal Failure(CRF) diagnosis for 4 years and receiving continuous ambulatory peritoneal dialysis (CAPD) treatment for 4 months. In peritoneal fluid, 1600/mm3 cells were detected and 70% of them were polymorphonuclear leukocytosis. The patient begun nonspesific antibiotherapy but no benefit was obtained after 12 days and peritoneal fluid bacterial cultures remained negative. Peritoneal smear was positive for Asid-fast basilli (AFB), and antituberculosis therapy was started with isoniazid, rifampicine, ethambutol and pyrazinamide. After 15 days his peritoneal fluid cell count was decreased and his symptoms were relieved. Peritoneal fluid tuberculosis culture was found positive. Conclusion Considering this case, we think that in patients with CAPD catheter and peritonitis; when peritoneal fluid leukocytes are high and PMNL are dominant, AFB and tuberculosis culture must be investigated besides bacterial culture routinely. | Introduction Tuberculosis continues to be a devastatingly important health problem in the world. In addition to pulmonary involvement, extrapulmonary involvement becomes an issue in most developing countries. Extrapulmonary tuberculosis, because of several factors, has greatly contributed to the total tuberculosis mortalities during the 20 th century [ 1 ]. Risk of tuberculosis has increased due to decreased immunity in uremic patients. Tuberculosis comes out extrapulmonary with a rate of 40 percent in these patients, and periton is involved in 6 percent of all cases [ 2 ]. The risk increases in hemodialisis patients within 12 months after the beginning of treatment [ 3 , 4 ]. Tuberculous (TB) peritonitis is an event rarely seen in continuous ambulatory peritoneal dialysis CAPD patients [ 5 ]. Our case is presented as a rare TB peritonitis event receiving CAPD treatment. Case Report The 36 year- old male patient, after receiving CAPD treatment for 4 months, consulted our clinic because of stomachache, diarrhea, nausea, vomiting and continous fever. The patient had been diagnosed with chronic renal deficiency and had been followed up with diagnosis of predialysis CRF for 4 years. The patient was referred to us because of his symptoms such as of nausea, vomiting, weakness, and a general condition of fatigue. Immediate care involved an urgent hemodialysis followed by CAPD and planning for renal replacement therapy. Through a physical examination, the patient's blood pressure was 110/70 mmHg. The general condition was bad and pulmonary sounds in the respiratory system were diminished slightly in lower zones. On CAPD catheter's entering segment, infections were not seen. In a laboratory investigation Hg was at 9.1gr/dl, Htc was at %27.6, WBC was at 5200/mm 3 , the platelet count was at 203000/mm 3 , and the erythrocyte sedimentation rate was at 22 mm/h. In biochemical findings, furthermore, serum creatinine was at 6.05 mgr/dl [ref. 0,5–1,4], urea nitrogen was at 38 mgr/dl [ref.5–20], protein was at 3.8gr/dl [ref:6–8.5], albumin was at 1.2 gr/dl [ref:3,5–5], and lactic dehidrogenase was at 565 iu/L. Serum sodium, potassium, glucose, bilirubin, alkaline phosphatase, aspartate and alanine aminotransferase, gamma-glutamyl transpeptidase, amylase, triglyseride, and cholesterol were normal. A coagulation factor protrombin time was found to be 18.7 sn. C-reactive protein was 9.54 mgr/dl. Bilateral costofrenic angles were blunted in posteroanterior pulmonary graphy. No parasites and cystes were found in fecal examination due to diarrhea. No pathogenic agent was detected in stool cultures. In peritoneal cell counting, 1600/mm 3 cell were detected and it was seen that 70 percent of these cells were polymorphonuclear leukocytosis (PMNL). The patient was given ceftazidime (IV), cephazol, and amikacin (intraperitoneal), but no benefit was noticed after 12 days of antibiotherapy and there was no growth in peritoneal fluid cultures. There were PMNL present but no microorganism could be detected. Acid-fast basilli (AFB) was found to be positive in the gram staining of peritoneal fluid in the remaining follow up periods, and the patient had begun antituberculosis therapy in fours(with isoniazid, rifampin, ethambutol and pyrazinamide). Tuberculin skin test was anergical. On the 15 th day of anti tbc therapy, peritoneal fluid cell count decreased to 300/mm 3 . Peritoneal fluid bacterial culture, blood cultures, throat culture and urine culture were negative but peritoneal fluid tbc culture was found to be positive, in Lowenstein- Jensen medium in 24 days. The patient was followed up with the treatment for recovery with an anti-tbc treatment. The peritoneal fluid of the patient was sent to be examined with Gram staining and Ziehl Neelsen staining. The peritoneal fluid was centrifuged at 3,000 × g for 15 minutes and the sediment was stained by Gram and Ziehl-Neelsen staining. The Gram staining showed PMNL presence but no microorganisms. The Ziehl-Neelsen staining(AFB) was positive. The peritoneal fluid was transferred to 10 ml sterile glass tube and centrifuged at 3,000 × g for 15 minutes. The concentrated sediment was inoculated onto Lowenstein Jensen (LJ) medium without prior decontamination. LJ medium was incubated at 37°C. Two specimens were later sent to be examined with Ziehl Neelsen staining on two different days. Both of them were detected to be positive for Ziehl Neelsen staining. LJ medium was examined for growth twice weekly for the first two weeks and once a week thereafter until the eighth week. After 24 days, the colonies were able to be seen on LJ medium. Positive growth was confirmed by Ziehl Neelsen staining. Discussion CRF increases the risk of tuberculosis. In patients receiving hemodialisis, the risk of tbc increases within twelve months after the occurrence of extrapulmonary tbc. The risk in these patients is ten times more for extrapulmonary tbc than in any other population. Peritoneal tuberculosis is rarely seen but remains a very important complication in CAPD patients[ 5 , 6 ]. Mortality is high in these patients [ 7 ]. There are literatures showing mortality rates as high as 15 percent [ 8 ]. Quantrill at al., in a TB peritonitis study with 8 cases, found bacterial peritonitis as a source of the patient's complaints [ 5 ]. It was reported that this patient' acute course was atypical with a predominance of neutrophils and low levels of protein in the peritoneal fluid [ 9 ]. In English literature the most common complaints of tbc peritonit are as follows: fever (78 percent), stomachache (92 percent), misty dialisat (90 percent) and PMNL are dominant in peritoneal fluids in 76 percent of the cases and in 73 percent of the cases AFB and culture are positive [ 8 ]. Abraham et al. have reported tbc peritonit in 4 of 155 CAPD patients and tuberculin test were found anergical in all patients [ 10 ]. In our case the tuberculin test result was found anergical as well. In a retrospective study made by Lui et al. pulmonary or extrapulmonary tbc was detected in 38 of 790 CAPD patients and they obtained benefits on the 7 th –57 th days of antituberculosis treatment (on average 30 day) [ 11 ]. We had experienced a recession in the peritonitis of the patient after 15th day of antituberculosis treatment. It was reported in the literature; that in tbc peritonitis treatment, removing peritoneal catheter has no apparent benefit and does not increase efficacy of the treatment [ 2 , 6 , 9 ]. Considering this case, we think that in patients with CAPD catheter and peritonitis; when peritoneal fluid leukocytes are high and PMNL are dominant, AFB and tuberculosis culture must be routinely investigated along with bacterial culture. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC523858.xml |
521083 | Single group study to evaluate the feasibility and complications of radiofrequency ablation and usefulness of post treatment position emission tomography in lung tumours | Background There is genuine need to develop interventional treatment options for management of lung tumors. Radiofrequency ablation (RFA) is one such alternative being promoted to treat lung tumors recently. Larger studies should help define RFA's further development. Furthermore fluorodeoxyglucose positron emission tomography (PET) has been reported to be an accurate indicator of treatment response in variety of tumors. This study focuses on the evaluating the feasibility of RFA and usefulness of PET scan in lung tumors after RFA procedure. Patients and methods Between November 1999 and May 2002, 50 patients with primary or metastasis pulmonary tumors underwent RFA procedure. The electrode was guided to the target areas using computerized tomography (CT). Tumors smaller than 3.5 cm were given single RFA, while tumors larger than 3.5 cm received RFA to multiple sites. Maximum 4 lesions or 6 target areas were treated during one operating procedure. Whole body and/or lung PET images were acquired; identical site CT images and chest X-ray were taken 1 week before and after RFA. Results Of the 50 patients, 17 had single lesions while rest had multiple lesions. Tumors smaller than 3.5 cm were completely dissipated after RFA. In tumors larger than 3.5 cm, the part within 3.5 cm diameter dissipated. While CT showed that tumor image became larger 1 to 2 weeks after RFA procedure. PET demonstrated tumor destruction in 70% cases, compared to 38% in CT. Conclusion The present study shows RFA to be safe and effective treatment option for lung tumors. PET is superior to CT in evaluation the effectiveness of RFA treatment shortly after the procedure. | Background Lung cancer continues to be the leading cause of cancer deaths in United States [ 1 ]. The overall prognosis of lung cancer is still dismal despite all current early detection and treatment efforts. Only about 20–25% of lung cancers can potentially be cured by surgery. The majority of patients presents with locally advanced or metastatic disease, and treatments essentially rely on external beam irradiation, chemotherapy or a combination of both [ 2 ]. Thus other interventional palliative treatment options have been developed for these lesions. Radiofrequency ablation (RFA), is an imaging-guided percutaneous ablative procedure, that has been suggested to be an effective treatment option for patients with non-small cell lung cancer (NSCLC) and metastatic disease who are not suitable candidates for surgery [ 3 , 4 ]. Guided by computed tomography (CT), physicians are able to localize the tumor and determine the optimal approach. During RFA, current passing through tissue from the active electrode leads to ion agitation and frictional heat generation. This leads to irreparable cellular damage and coagulation necrosis [ 5 ]. Recently a number of studies reported its application in malignant lung tumours. Accurate assessment of treatment response remains one of the major problems. PET has been reported to be an accurate indicator of treatment response in variety of tumors [ 13 - 17 ]. However, its use has been limited to evaluating disease stage in lung tumors [ 18 - 24 ]. PET imaging, provides proliferation and metabolism information, is sensitive and specific to diagnose malignant lesions from benign. Coleman and colleagues has provided substantial information in evaluating the role of PET in management of lung cancers [ 21 - 23 ]. In this report we focus on evaluating the feasibility of RFA, its complication and on evaluating the role of PET on RFA response in lung tumors. Patients and methods Between November 1999 and May 2002, 50 patients with either primary or metastatic lung tumors were enrolled in to a prospective single group trial. Patient characteristics are detailed in Table 1 . Patients with bleeding potentials or serious heart, liver and renal failures were excluded. Antibiotics and medicines for prevention of bleeding were given regularly. Every patient underwent a chest Flurodeoxyhlucose postron emission (PET) and CT scan before procedure. Table 1 Patient characteristics Patients (n = 50) Patient characteristic No % Age, years Median 51 Range 35–74 Sex Male 32 64 Female 18 36 Origins Primary lung tumors 23 46 Metastases from breast 13 26 Metastases from colon 9 18 Metastases from other places 5 10 No. of patient with lesions Single lesion 17 34 Multiple lesions 33 66 Total lesions received RFA 120 Patients received a chest X-ray and CT for preoperative evaluation and a repeat scan after RFA procedure. A PET scan was performed one week after the treatment. The Radiofrequency ablation was carried out using RF-2000 generator and related software purchased from Radio Therapeutics Corporation, USA; PET imaging was done using an Advance 2 Scanner (General Electric Medical Systems, WI, USA). Patients received general anesthesia along with local infiltration of Lidocaine. The electrodes were directed to target areas during RFA procedure using CT scan. The initial power applied was 50 W, which was subsequently increased to a maximum 90 W over several minutes. RFA continued for 5 to 15 min until roll off was achieved, which continued for 2 min to stop. Tumors smaller than 3.5 cm were given full heating energy only once, while tumors larger than 3.5 cm received multiple RFA to different areas. Maximum 4 lesions or 6 target areas were treated during one procedure. One to two weeks after the procedure and a 4 hour fast, patients were taken for PET scan. They were made to rest for 15 min, and then received 18 F-FDG 296 MBq – 440 MBq (8 mCi -12 mCi) intravenously. After another period of rest lasting for 45–60 min, the whole body and/or lung images was acquired by PET scanner. PET was also acquired at 5–8 bed positions, typically from the base of skull to the mid thigh, which was identical to the CT protocol used in the present study. The complications of the treatment are detailed in table 2 and results are summarized in table 3 . Table 2 Complications of Radiofrequency ablation Patients (n = 50) Complication No % Fever 10 20 Congested pneumonia 6 12 Pneumothorax 9 18 Hemothorax 1 2 Table 3 Early effectiveness of RFA by various techniques Tumor destruction demonstrated Technique No % All 50 patients received PET 35 70 CT 19 38 X-ray 13 26 Results After RFA procedure a number of complications were seen. Fever and/or congested pneumonia were commonest complications seen in 32% of patients; however, they were cured in a week with antibiotics treatment. Pneumothorax occurred during procedures in 18% and the patients were treated with aspiration. Five of these had small pneumothorax that did not require and treatment. One patient had hemothorax which required intercostals drainage (ICD) which was removed 2 days later. These were no life threatening events or deaths. Post procedural PET demonstrated the effectiveness of RFA on lung tumors. Tumors smaller than 3.5 cm showed complete response after RFA (Figure 1 ). In tumors larger than 3.5 cm, the part within 3.5 cm diameter dissipated, while the part outside this 3.5 cm area remained (Figure 2 ). Damage to the normal tissue outside the tumor was not extensive in any cause. Figure 1 PET images taken before and after RFA treatments: coronal (A, B), and Sagittal (C, D) views of PET scans of lung cancer. (A, C) were taken before RFA treatment; (B, D) were taken two weeks after RFA treatment. Figure 2 PET images taken before and after RFA treatments. This patient had a tumor size larger than 3.5 cm. PET scans were taken 1 week before (A) and 2 weeks after (B) RFA treatment. The Chest X-ray and CT showed that tumor image became larger 1 to 2 weeks after RFA procedure (Figure 3 ). These may result from partial tissue damages, bleeding, acute inflammation or pneumonia, and support the routing use of antibiotics and haemostatic drugs after RFA. The tumor destruction was picked up by PET much effectively when compared to CT scan or chest X-ray. Figure 3 CT images taken before and after RFA treatments. The same patient PET images were shown as Figure 1. (A) was taken before RFA treatment, (B) was taken 2 week after RFA treatment. Discussion Since RFA ablates lung tumors directly and locally, marginal tissues surrounding the tumor are frequently partially damaged leading to occasional pneumonia. It is difficult for regular CT and/or chest X-rays to discriminate pathological-physiological tissue damage and fibrillation from the treatment effect of RFA. PET on the other hand provides information on functional and metabolic activity anatomically, and is the only available technique which can specifically diagnose tumors or necrosis after surgery and radiotherapy effectively [28]. Our experience too proves that PET is particularly superior to CT in its ability to evaluate the effectiveness of RFA treatment early after therapy. RFA is a relatively noninvasive, well-tolerated approach. It could destruct tumor completely within the effective diameter while avoiding the surgery, side effects of radiotherapy and toxicity of high dose chemotherapy. Our observations suggest that RFA can kill lung tumors smaller than 3.5 cm after a single RFA procedure. The effect of RFA appears to be limited within 3.5 cm diameter area with the current instruments. However, this also suggests that RFA may not damage the normal tissues surrounding the small tumors. The malignant lesions dissipated in 1 to 2 weeks, while the surrounding tissue stayed intact. While at this period regular chest CT and chest X-ray may show enlarged lesion images. This is in agreement with other reports. With improvements in technology, RFA in combination with other options may further reduce the morbidity and mortality of cancer deaths [ 11 ]. Though complications do occur, they are usually curable. RFA results in a higher rate of complete necrosis and requires fewer treatment cycles compared to traditional chemotherapy or radiotherapy. Besides CT guidance help to localize the tumor and determine the optimal approach further optimizes specific of targeting the tumor. For patients with non-small cell lung malignancy who are not candidates for surgery owing to poor cardio respiratory reserve, RFA alone or followed by conventional radiation therapy or chemotherapy may prove to be a treatment option [ 11 ]. For patients with metastatic disease, RFA may be suitable for treatment of a small tumor or reduce symptoms caused by large tumor burden. This technique can be used as a primary technique or in conjunction with other interventional procedures [ 11 ]. Further randomized controlled trials comparing RFA with conventional palliative treatment are needed before RFA can be accepted as a routine treatment modality. Survival of patient and quality of life issues too need be addressed. Conclusions Despite inherent deficiency of trial design our single group study clearly demonstrates that RFA can be an effective treatment option for lung tumors. Unlike other interventional techniques, RFA provide controlled regions of coagulation necrosis with a single application to an area with 3.5 cm diameter. RFA may cure small lung tumor, reduce tumor burden in larger lesions and may be combined with external beam radiation and/or systemic chemotherapy for further improvements. PET provides functional and metabolic activity anatomically and is particularly superior to CT in evaluation the effectiveness shortly after RFA procedure. Absence of follow-up information and randomization in the current study are two major fallacies which need to be addressed in subsequent studies. Authors' contributions SJK is the leading physician and drafted the manuscript. RL, WL, HW, XZ, YM all participated in the study, patient management, literature search and preparation of manuscript. All authors have read and approved the final version of the manuscript. Competing interests None declared. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521083.xml |
406400 | Open Access: A PLoS for Education | Teaching -- and testing -- students creatively is a challenge, but new public databases and more accessible literature are now helping to develop the critical thinking skills of students | The next generation of life scientists are currently undergraduates—and the success of this generation depends upon the quality of the education they receive. It is clear the expectations for undergraduate education are changing ( Collins et al. 2003 ). When the National Research Council published its recommendations for changing the undergraduate training of future life scientists, the BIO2010 report, access to student-based research was a primary recommendation: “Colleges and universities should provide all students with opportunities to become engaged in research …” ( National Research Council 2003 ). As every investigator knows, research begins in the literature, not in the laboratory. Therefore, an unstated assumption of the BIO2010 report was that students need to have unencumbered access to the research literature in order to engage in research and become scientific leaders in the 21st century. Early in my teaching career, I discussed graduate student preparation with a colleague at MIT. He said new graduate students knew about the different methods, they could even recite fine definitions—but if you asked them which method would be best to answer a particular question, they were uncertain. This reinforced my attitude towards teaching and testing. I realized that teaching science to students should be modeled on the way all scientists learn new information: in the context of an interesting question and on a need-to-know basis. This new style of teaching, “applied education,” would require me to reorganize reading materials for students, since most textbooks are written by someone who already knows all the information and has organized it accordingly. For example, describing membrane structure, protein structure, and signal transduction in Chapters 5, 12, and 15, respectively (spanning 227 pages) is not helpful for most students. It makes more sense to cover these three topics in close succession. Gradually, I converted all my courses over to this “applied education” format in which students were learning new information the same way all other scientists do. I began by asking questions that could be answered by learning the information provided by textbooks or the literature. With time, I realized that published research papers are ideal teaching tools because they cover information in the context of an interesting question and new material is presented as needed. This led me to collect series of related papers to create my own course materials (see www.bio.davidson.edu/courses/Molbio/Publicschedule.html#anchor99574051 ). So, for example, in my classes students first read the elegant paper by Munro and Pelham (1987) that uncovered the tetrapeptide lysine–aspartic acid–glutamic acid–leucine (KDEL) retention signal for proteins destined to remain in the endoplasmic reticulum lumen. Then, students read four additional papers, one of which is composed of weak data and overinterpreted analysis. Through this series of papers, students learn to trust their own assessment of the data rather than the authors': this is a very substantial improvement in student thinking and in their attitude towards the literature. I do not emphasize the particular details of these paper, but I do want the students to gain higher-order thinking skills. Therefore, my tests consist of figures from research papers that the students have never seen before. They are asked to interpret the figures as they appear in the papers and/or to design new experiments to answer a new question, given what they have learned from the published figure. Testing them in this way, students very quickly understand that memorizing details is not productive, but learning how to read scientific literature and design well-controlled experiments is much more rewarding (see www.bio.davidson.edu/courses/Molbio/molecular.html#2003exams ). Based on this success, I have designed my genomics course on the “applied education” principle (see below; see also www.bio.davidson.edu/genomics ). Access to Information Changes Education When I was a graduate student (in the late 1980s and early 1990s), PubMed was restricted to those institutions that could afford the subscription fee; now PubMed is freely available to all who have Internet access. This change in access to PubMed has significantly improved undergraduate training by providing students with the opportunities to do literature searches for their lab reports, papers, seminars, and of course original research. Free access to information in the life sciences has continued to evolve with the newest phenomenon in publishing—open-access journals. PubMed Central ( http://www.pubmedcentral.nih.gov/ ) is a rich repository of and portal to open access articles, BioMed Central ( http://www.biomedcentral.com/ ) publishes a growing number of open-access journals, and there are a few new open-access education journals such as Cell Biology Education ( http://www.cellbioed.org ) and the Journal of Undergraduate Neuroscience Education ( http://www.funjournal.org ). As the newest player in the open-access arena, PLoS Biology has further enriched the growing espritdes-corps of publishing and has already improved undergraduate education. My students now have equal access to a growing portion of the literature that Nobel laureates and investigators at wealthy institutions enjoy. Interestingly, the push towards open access has led many subscription-based journals to permit “free access” two to 12 months after publication. These time-delayed free-access journals are helpful for course adjustments in the subsequent academic year, but not the current semester. Unfortunately, owing to the high cost of subscriptions for many journals, the library at my institution (like many other libraries) is forced to make difficult choices about which journals we can afford. The number of journal subscriptions goes down in proportion to the rise of subscription costs, but fortunately this loss is being offset by the creation of new open-access journals. The Promise of the Internet I have been teaching undergraduates since 1993 and have noticed a trend in the way I teach—increasingly, I have provided research papers to my students so they can learn to read those papers and improve their critical thinking skills. One reason for my increased use of research papers is the development of PDFs. When I first started using journal articles in my molecular biology course, the class had to meet in the library so we could pass around the bulky bound volumes to detect the important subtleties often lost in photocopied versions of figures. Later, I learned how to scan the figures and generate Web pages so that I could project the images in class and so that students could print laser-quality versions of papers (see http://www.bio.davidson.edu/molecular ). Now I use PDF files for students to print and for me to display in class with no loss of information due to reformatting or resolution problems ( Figure 1 ). Figure 1 Comparison of Published and Photocopied Figures Example of an image that, when seen in color (A), is rich with information; much of this information is lost when it is photocopied by students (B), as when the original is held on reserve in the library, as is required for subscription-based journals, or is provided via interlibrary loan. This image of a developing fly embryo was labeled to reveal bands of differentially expressed proteins, with HAIRY in red, KRüPPEL in green, and GIANT in blue. (Image courtesy of Stephen W. Paddock, Jim Langeland, and Sean Carroll at the University of Wisconsin–Madison.) With my increased confidence from using research papers in my molecular biology class, I began experimenting with research papers for my introductory students. First-year students are not ready to critically evaluate complex data, but they are beginning their first forays into reading review articles and occasionally original research papers. When introductory students make presentations of their findings in laboratory courses, increasing numbers are utilizing PubMed and PDF reprints when they are available. Students have been reading primary research papers since well before PDF files became available, but the increased access to papers online and the improved quality of the format has significantly enhanced the use of research and review papers in the undergraduate curriculum. It is common for students in upper-level lecture and lab courses to read papers ( DebBurman 2002 ; Hall and Harrington 2003 ; Kitchen et al. 2003 ; Mulnix 2003 ), and seminar courses are usually dominated by student presentations of literature ( Wright and Boggs 2002 ; Hales 2003 ; Lom 2003 ). It is worth noting that most colleges and universities are being told to reduce expenditures, and one frequent target of money-saving measures is the ever-increasing costs of library journal subscriptions. This fiscal reality will erode the pedagogical gains made by faculty who are already meeting one of the goals of the BIO2010 report by immersing students in the research literature. However, open-access journals are proving to be virtual oases in a desert of pay-per-view journals that are available on a sliding scale that favors the richest and biggest institutions. Using Open-Access Resources for Creative Teaching … During the past three years, I have taught an undergraduate course in genomics ( www.bio.davidson.edu/genomics ) in which I capitalize on a confluence of two trends in the field: public domain databases and open-access journals ( Campbell 2003 ). In my genomics class, students have three assignments for which they are required to mine databases for sequence, transcriptome, and proteome information (see www.bio.davidson.edu/courses/genomics/2003/cain/home.html ). But genomics courses are not the only beneficiaries, since other classes at many institutions (e.g., introductory biology, biochemistry, cell development, genetics, microbiology, molecular biology [see http://www.bio.davidson.edu/courses/Molbio/standardsHP.html#anchor78181983 ], and neuroscience) require students to mine public domain databases ( Dyer and LeBlanc 2002 ; Honts 2003 ). This year, we introduced genome database searching to our introductory biology students (see www.bio.davidson.edu/people/macampbell/Hope/DQ/DQ9.html and www.bio.davidson.edu/people/macampbell/Hope/DQ/DQ10.html ). First-year students use Genome Browser and BLAST to determine the molecular causes of cystic fibrosis and Huntington disease, respectively. The benefit of public databases and open-access literature to educators is obvious and immediate. Images can be used in lectures, and papers can be distributed easily and on short notice for class use. There is no need to worry about limited access due to subscription costs nor an obligation to obtain copyright permission from publishers, which is a bothersome and sometimes expensive process for busy faculty members. By reducing nonproductive busy work for faculty, open-access journals have already created an environment that is improving undergraduate education today with long-term benefits in creating research-ready graduate students. Students who are exposed to publicly available literature through their coursework often develop an expectation that all research papers will be freely available to them from any computer and become frustrated if they do not have access to all the journal articles they want and need to read. Increasingly, I have students sending me PDF files of open-access journal articles they have read and want to share with me. Who would have guessed that free access to journals would result in students mining the literature for relevant papers and sending them to their instructors for consideration? In addition to papers related to their own classes and research, students also enjoy learning about “hot topics” from scientific publications and those stories that quickly reach the popular press. Examples include the use of DNA microarrays and sequencing to identify the causative agent for SARS ( Wang et al. 2003 ) and a good review article of small inhibitory RNA ( Dillin 2003 ). Two common educational goals are to encourage students to become skeptical of unsubstantiated claims and to enable students to evaluate data critically. One way to accomplish these goals is to capitalize on the natural curiosity of students and ask them to compare topics in the popular press to that in the scientific literature (see http://www.bio.davidson.edu/courses/genomics/2003/poulton/p21.html ). Open-access journals make these two educational goals much more feasible because students can utilize current findings immediately without having to wait for interlibrary loans, which can take up to two weeks, can cost up to $20 per article, and can result in poor-quality black-and-white photocopies. … and for Thought-Provoking Testing If we want students to achieve higher levels of thinking ( Bloom et al. 1956 ), we need to model our courses so students can learn by examples and are rewarded for learning to critically evaluate data and for inspecting evidence before believing claims made by authors ( Brill and Yarden 2003 ). Students quickly figure out what intellectual behaviors are rewarded in exams. If exam questions simply require students to regurgitate factoids, then higher levels of thinking are unlikely to be demonstrated by students. It is difficult to create good exam questions that cover the course material and reward students who have learned to read critically and to interpret data. Over the last few years, increasingly I have turned to current literature to find raw data for my exam questions. For example, for my genomics class in Fall 2003, I used a paper published in PLoS Biology that utilized DNA microarrays to analyze the life cycle of malaria-causing Plasmodium ( Bozdech et al. 2003 ). I asked students to interpret several figures, using their own words ( Figure 2 ). Owing to my choosing to use an open-access journal, my students also had full access to the supporting information, which two students utilized to enhance their answers. For this question, these two produced answers that were better than mine. Another exam question required students to mine a database associated with the Bozdech paper (see http://malaria.ucsf.edu/index.php ). Students were asked to combine what they learned from the paper and the course and choose new proteins (in addition to the ones described in Bozdech et al. [2003] ) that would make good candidates for vaccines based on the timing of gene transcription. In order to answer this question, students performed the first steps in real research, which rewards students for learning higher-order thinking skills. Figure 2 Example of Student's Data Mining for Exam Question Figure 2 from Bozdech et al. (2003) showing the gene expression profiles for 12 groups of genes expressed at different stages of Plasmodium life cycle inside red blood cells. Genomics students were asked to summarize this figure as a part of a take-home exam. At the end of their exam, students were given an opportunity for extra credit points (a maximum of three points out of 100 available on the exam) if they provided constructive criticism directly to the database curators. About 70% of the students sent comments, including this one: “In recently using your database, I found it difficult to search the Plasmodium gene expression data with multiple constraints. For example, it would be helpful if there were a way to identify all the genes within a certain functional group that fell within certain time or amplitude constraints. Is this possible in this database?” The curators very professionally responded to the students' suggestions, which resulted in three new search capacities being added to the database, as can be seen on the left side of the main page (see http://malaria.ucsf.edu/index.php ). As a result of these professional interactions, students became participants in a community of scholars, interacting with investigators at the University of California, San Francisco, while taking their exams. The use of open-access journals for teaching and testing has already improved my courses. I can provide exam questions that are more interesting, more educational, and more current. Furthermore, I accomplish two tasks simultaneously: I keep abreast of new developments in my field and I write exam questions. But what do students think? While I have not formally assessed student attitudes, I have collected information from end-of-semester course evaluations, including the following comments: “One of the best parts of the entire course for me were the exams. The exams really gave me an opportunity to show how I could work through real problems. This class definitely increased my critical thinking skills. Each test presented me with new ideas and problems to work through. I enjoyed the idea that each exam would be a learning experience.” The Future Teaching is a lot like raising children. Like parents, teachers provide learning opportunities in part by modeling the behavior we want our students to learn. By choosing the most current literature as testing material, my students realize that I read the literature to stay current in my field and that there are always new opportunities to learn, analyze, and design experiments, etc. By my choosing open-access papers such as those published in PLoS Biology , my students benefit from free access to published research results. Free access to research literature enhances student learning and helps produce the next generation of graduate students, who are then better trained. Open-access publishing provides the right mix of benefits for educators and students alike. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC406400.xml |
406399 | The Mre11 Protein Is Necessary for DNA Damage Response | xx | With billions of cells in the adult human body, all replicating and dividing in an environment laden with toxins, radiation, and free radicals, a certain amount of DNA damage is guaranteed to occur. Fortunately, all organisms have built-in checkpoints throughout the cell cycle that prevent such mistakes from propagating. At the G1 checkpoint during cell division, for example, molecules survey nuclear DNA for errors and breaks before the cell is deemed fit to undergo S phase, the DNA replication stage. If damage is found, enzymes either work to repair it or, in some cases, trigger programmed cell death, or apoptosis. But when checkpoints fail, and DNA damage is left unrepaired, disease such as cancer can result. A better understanding of these events, as provided, for example, by Vincenzo Costanzo and colleagues in this issue, will consequently lead to a better understanding of the mechanisms that give rise to cancer. Model for Mre11 complex bridging DNA molecules A serious form of DNA damage, called a double strand break (DSB), cuts the helix clean through—a far worse scenario than if just one strand slips free. In response to a DSB, the cell recruits a signaling protein called ATM and a three-protein complex called MRN, whose components selectively bind to broken DNA ends. A malfunction of this signal and repair pathway is dire. People who suffer from the genetic disease ataxia-telangiectasia (A-T) lack a functioning ATM molecule and therefore cannot properly handle DSBs or successfully navigate the G1 checkpoint. This condition leads to a host of problems, including abnormal chromosomes, deficient immune function, and a predisposition to cancer. A-T-like disease (ATLD), another rare genetic condition, has very similar symptoms. The only difference is that the protein missing is Mre11, a subunit of MRN. While recent work on the cellular level has indicated that MRN activates ATM, the biochemical relationship between these proteins has yet to be fully understood. Studying these two molecules using traditional biochemical assays is difficult because knocking out the activity of these proteins is lethal to many cells. Costanzo and colleagues used a novel test system of cell-free frog extracts and found that Mre11 is necessary for both ATM activation and for the formation of large protein–DNA complexes apparently responsible for triggering the cascade of signaling molecules underlying the DNA damage response at the G1 checkpoint. The frog extract system allowed the team to manipulate the presence or absence of Mre11 and accurately measure the response triggered by the addition of fragmented bits of DNA (simulating naturally occurring DSBs). As predicted, without a functional Mre11 protein, ATM was not activated and there was no response. By simply adding the protein Mre11 back to the mixture, the damage response was restored. But when the researchers added a mutant form of Mre11, still capable of performing its essential tasks in another stage of the cell cycle—DNA replication—the G1 damage response remained suppressed. This mutant form of the Mre11 protein lacks the C-terminal, or DNA-binding, end. Costanzo and colleagues also found that this DNA-binding end is required for the assembly of DNA–ATM–MRN complexes in the presence of fragmented DNA and seems to direct the entire damage response. This work helps to explain the similarity between patients with A-T and those with ATLD, and hints at the formation of a large “signaling” complex that helps to orchestrate the crucial response to DBSs in DNA. | /Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC406399.xml |
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