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+    "id": "PMC2527550",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527550\nAUTHORS: T Eoin West, Robert K Ernst, Malinka J Jansson-Hutson, Shawn J Skerrett\n\nABSTRACT:\nBackgroundMelioidosis, a lethal tropical infection that is endemic in southeast Asia and northern Australia, is caused by the saprophytic Gram-negative bacterium Burkholderia pseudomallei. Overall mortality approaches 40% yet little is known about mechanisms of host defense. Toll-like receptors (TLRs) are host transmembrane receptors that recognize conserved pathogen molecular patterns and induce an inflammatory response. The lipopolysaccharide (LPS) of Gram-negative bacteria is a potent inducer of the host innate immune system. TLR4, in association with MD-2, is the archetype receptor for LPS although B. pseudomallei LPS has been previously identified as a TLR2 agonist. We examined TLR signaling induced by B. pseudomallei, B. pseudomallei LPS, and B. pseudomallei lipid A using gain-of-function transfection assays of NF-κB activation and studies of TLR-deficient macrophages.ResultsIn HEK293 cells transfected with murine or human TLRs, CD14, and MD-2, heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6) and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B. pseudomallei augmented TNF-α and MIP-2 production whereas in TLR4-/- cells, TNF-α, MIP-2, and IL-10 production was reduced. Cytokine production by macrophages stimulated with B. pseudomallei LPS or lipid A was entirely dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule MyD88 strongly regulated TNF-α production in response to heat-killed B. pseudomallei.ConclusionB. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B. pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or lipid A, cytokine production is augmented. Other MyD88-dependent signaling pathways may also be important in the host response to B. pseudomallei infection. These findings provide new insights into critical mechanisms of host defense in melioidosis.\n\nBODY:\nBackgroundMelioidosis is an endemic and poorly understood infectious disease in much of the tropical world; it is particularly prevalent in east Asia and northern Australia. The disease accounts for 20% of community-acquired sepsis in parts of northeast Thailand. Despite antibiotic treatment, mortality rates approach 40% [1]. The causative organism, Burkholderia pseudomallei (Bp), is a Gram-negative environmental saprophyte. Aerosol or transcutaneous infection results in an extensive range of disease – from chronic, relapsing illness with abscess formation to fulminant pneumonia and septicemia [2]. The lung is the most commonly affected organ. Concern about the use of Bp as a bioweapon has led to its classification as a CDC Category B pathogen. While there are several known predisposing factors to clinical infection, such as diabetes, renal or liver disease, alcoholism, or immunosuppression [3], relatively little is known about mechanisms of host susceptibility.Innate immune signaling mechanisms comprise the front line of host defense against infection. Toll-like receptors (TLRs) are transmembrane receptors in the IL-1 receptor superfamily that are activated by conserved pathogen-associated molecular patterns and result in nuclear transcription factor (NF)-κB translocation and induction of a pro-inflammatory response [4]. TLR2, in tandem with TLR1 or TLR6, is activated by bacterial cell wall lipopeptides and peptidoglycan. TLR4, in association with proteins CD14 and MD-2, recognizes the lipid A component of lipopolysaccharide (LPS) of most Gram-negative organisms. The importance of TLR2 and TLR4 in pulmonary host defense has been well established [5-9].As a Gram-negative bacterium, Bp has putative ligands for both TLR2 and TLR4 [4]. Wiersinga et al. found increased TLR2 and TLR4 expression and mRNA levels in monocytes and granulocytes of humans with melioidosis as well as activation of these TLRs by Bp in vitro [10]. Wiersinga et al. also reported a protective phenotype in TLR2-deficient mice infected intranasally with Bp. However, they unexpectedly observed that Bp LPS activates TLR2, not TLR4, in transfected HEK293 cells.In this study, we evaluated the roles of TLR2 and TLR4 in melioidosis using TLR transfection constructs as well as bone marrow-derived macrophages from TLR-deficient mice. We report here that heat-killed Bp activates TLR2 in conjunction with TLR1 or TLR6, as well as TLR4. We also show that cytokine production induced by stimulation of primary cells with heat-killed Bp is dependent on TLR4 but that cytokine release is augmented in the absence of TLR2. We further demonstrate that Bp LPS and Bp lipid A are ligands for TLR4, not TLR2.MethodsPreparation of bacteriaBp BP-1, a clinical isolate cultured from a liver abscess of a Vietnamese patient presenting for medical care in Washington state, was grown for 19 hours at 37°C in LB broth. Bacteria were washed twice in sterile PBS and resuspended in PBS. The bacterial slurry was then heat-killed for 60 minutes at 65°C. Bacterial concentration and confirmation of successful killing was determined by quantitative culture on LB agar of the washed and heat-killed slurries, respectively.LPS and lipid A isolation and purificationLarge-scale BP-1 LPS preparations were isolated using a hot phenol/water extraction method after growth of BP-1 in LB supplemented with 1 mM MgCl2 at 37°C [11]. Subsequently, LPS was treated with RNase A, DNase I and proteinase K to ensure purity from contaminating nucleic acids and proteins [12]. Reference strain Bp K96243 LPS was kindly provided by Donald Woods at the University of Calgary. BP-1 and K96243 LPS samples were additionally extracted to remove contaminating phospholipids [13] and TLR2 contaminating proteins [14,15]. LPS preparations were determined to have less than 1% contaminating protein as determined by BCA reaction (Pierce, Rockford, IL). Lipid A was isolated after hydrolysis in 1% SDS at pH 4.5 as described [16]. Briefly, 500 μl of 1% SDS in 10 mM Na-acetate, pH 4.5 was added to a lyophilized sample. Samples were incubated at 100°C for 1 hour, frozen, and lyophilized. The dried pellets were resuspended in 100 μL of water and 1 mL of acidified ethanol (100 μL 4 N HCl in 20 mL 95% EtOH). Samples were centrifuged at 5,000 rpm for five minutes. The lipid A pellet was further washed three times in 1 mL of 95% EtOH. The entire series of washes was repeated twice. Samples were resuspended in 500 μL of water, frozen on dry ice and lyophilized. Before use, samples were resuspended in sterile water. Finally, negative ion matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) experiments were performed for the analysis of lipid A preparations to profile structures present in these preparations. Lyophilized lipid A was dissolved with 10 μL 5-chloro-2-mercaptobenzothiazole (CMBT) (Sigma-Aldrich, St. Louis, MO) MALDI matrix in chloroform/methanol, 1:1 (v/v), and then applied (1 μL) onto the sample plate. All MALDI-TOF experiments were performed using a Bruker Autoflex II MALDI-TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA). Each spectrum was an average of 200 shots. ES Tuning Mix (Agilent, Palo Alto, CA) was used to calibrate the MALDI-TOF MS.HEK293 transfections and stimulationsHEK293 cells were cultured in a 96 well flat-bottomed tissue culture plate at ~5 × 104 cells/well in DMEM plus 10% FBS. The following day cells were transiently transfected with 5 μL of transfection reagent comprised of a 1:1 mix of 0.25 M CaCl2 containing 2 × BBS (50 mM BES, 280 mM NaCl, and 1.5 mM NaH2PO4) and the following DNA: NF-κB-dependent firefly ELAM luciferase and control β-actin-dependent Renilla luciferase; and human or murine CD14, MD-2 (except as noted), with either TLR2, TLR2 and TLR1, TLR2 and TLR6, or TLR4 [17]. Two different human TLR1 plasmids were transfected in conjunction with TLR2, one with G at base pair 1805 (which yields lower responses to Pam3CSK4) and one with T at this position (which yields higher responses to Pam3CSK4) [7]. DNA was added in the following amounts to each well: hu- or muMD2 0.0025 μg, hu- or muCD-14 0.0025 μg, ELAM luciferase 0.01 μg, and Renilla luciferase 0.0003 μg. When transfected alone, the following amounts of DNA were added: hu- or muTLR2 0.0025 μg, huTLR4 0.0025 μg, or muTLR4 0.0003 μg. When co-transfected with TLR1 or TLR6, huTLR2 0.00125 μg or muTLR2 0.0025 μg was used in addition to huTLR1 0.00125 μg or muTLR1 0.025 μg; or huTLR6 0.0125 μg or muTLR6 0.0025 μg. All transfections were normalized to 0.05 μg total DNA with the addition of empty vector. Transfected cells were washed once after four hours and were stimulated the following day with experimental ligands, non-specific stimulus recombinant human IL-1β (Pierce Endogen, Rockford, IL), control TLR4 ligand ultrapure Escherichia coli 0111:B4 LPS (Invivogen, San Diego, CA), or control TLR2 ligand Pam3CSK4 (Invivogen and EMC Microcollections, Tuebingen, Germany). After four hours, cells were lysed with passive lysis buffer (Promega, Madison, WI) and NF-κB activation was determined in 10 μL of lysate by the ratio of firefly to Renilla luciferase light emission using the Dual Luciferase Reporter System (Promega, Madison, WI).AnimalsSpecific-pathogen-free C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). TLR2-/-, TLR4-/-, TLR2/4-/-, and MyD88-/- mice backcrossed at least six generations to C57BL/6 mice were obtained from Dr. Chris Wilson at University of Washington. All animals were housed in laminar flow cages and were permitted ad lib access to sterile food and water. Euthanasia was accomplished with intraperitoneal pentobarbital followed by exsanguination from cardiac puncture prior to bone marrow harvest. The Institutional Animal Care and Use Committee of the University of Washington approved all experimental procedures.Bone marrow-derived macrophage stimulationsFemurs and tibias were harvested under sterile conditions from wild type, TLR-deficient, and MyD88-/- mice. Marrow was flushed out using a 26 gauge needle through a 0.2 μm strainer and cultured in Petri dishes in RPMI media supplemented with 1% L-glutamine, 1% penicillin-streptomycin, 10% fetal bovine serum and 20% L929 cell conditioned media at 37°C under 5% CO2 for 5–10 days to allow macrophages to predominate. The monolayer was washed twice with HBSS or media, and macrophages resuspended in RPMI media supplemented with 1% L-glutamine, 1% HEPES, and 10% fetal bovine serum. Cells were added to a 96 well flat-bottomed tissue culture plate at 3 × 104 to 2 × 105 cells/well, depending on the experiment. The following day, cells were stimulated with experimental ligands, control TLR4 ligand ultrapure E. coli 0111:B4 LPS (Invivogen, San Diego, CA), or control TLR2 ligand Pam3CSK4 (Invivogen) in fresh media added to each well. After stimulation for 24 hours, supernatants were removed and stored at -80°C until assayed. TNF-α, MIP-2, and IL-10 were quantified in the supernatants using DuoSet ELISA (R&D Systems, Minneapolis, MN).Statistical analysesComparisons between two groups of normally distributed data were performed using the t test. Comparisons between three or more groups of normally distributed data were performed with analysis of variance followed by the Bonferroni post-test between groups. Statistical testing was undertaken using Stata v9.0 (StataCorp, College Station, TX). Statistical significance was declared for two-tailed p < 0.05.ResultsIn order to identify TLRs that recognize Bp, HEK293 cells transiently transfected with plasmids expressing murine TLR2, TLR2/1, TLR2/6, or TLR4, and co-receptors CD14 and MD-2 were stimulated with a heat-killed Bp clinical isolate BP-1 (Figure 1A). Using an NF-κB-dependent luciferase reporter assay, strong Bp-induced NF-κB activation was detected in TLR2- and TLR4-transfected cells with augmentation of the TLR2-dependent signal in the presence of TLR1 or TLR6. To determine whether similar signaling is triggered by human receptors in response to Bp, HEK293 cells transfected with plasmids expressing human TLRs were also stimulated with Bp (Figure 1B). TLR2- and TLR4-mediated NF-κB activation was observed in a largely dose dependent fashion. Both TLR1 and TLR6 augmented the TLR2-mediated response. We also transfected a hypo-responsive variant human TLR1 plasmid in addition to TLR2 in this system and observed little amplification of the TLR2-dependent signal upon stimulation with Bp (data not shown). Because MD-2 is a necessary molecule for TLR4 signaling, human TLR4 transfection was repeated without co-transfecting MD-2. In the absence of MD-2, a TLR4-mediated signal in response to Bp was not detected (Figure 1C) although TLR2-mediated signaling was not altered (data not shown).Figure 1B. pseudomallei activates TLR2 and TLR4. HEK293 cells were transiently transfected with (A) murine or (B) human TLR2, TLR2/1, TLR2/6, or TLR4; co-receptors CD14 and MD-2; and NF-κB-dependent firefly ELAM luciferase and control β-actin-dependent Renilla luciferase. In (C) HEK293 cells were transiently transfected with human TLR4 and co-receptor CD14 with or without MD-2; and firefly ELAM and Renilla luciferases. Cells were stimulated with media alone, (B) IL-1β 20 ng/mL, (A & B) Pam3CSK4 100 ng/mL, E. coli 0111:B4 LPS 10 ng/mL, or heat-killed BP-1 at various concentrations in CFU/mL. NF-κB activation was measured by light emission (relative light units). In (A&B) data plotted are means ± standard deviations of triplicate conditions. In (C) the means ± standard deviations of triplicate human TLR4-mediated relative light units (normalized to mean empty vector values) are plotted from parallel experiments with or without co-transfection of MD-2. For (A & B) * indicates p < 0.05 and § indicates p = 0.001 compared with empty vector stimulated with the same ligand, and † indicates p < 0.05 compared with TLR2 stimulated with the same ligand using analysis of variance followed by the Bonferroni post-test. Other comparisons are not shown for clarity. The data in (A) represent one of two independently performed experiments. The data in (B) represent one of three independently performed experiments, but the data displayed, in contrast to the two other experiments, show the response to a hyper-responding variant TLR1 plasmid. The data in (C) represent one of three independently performed experiments.Since robust TLR4-dependent NF-κB activation was observed using heat-killed Bp, we sought to identify which Bp ligand activates TLR4 signaling. As Bp LPS was the most likely candidate, LPS was isolated and purified from the Bp clinical isolate BP-1. LPS from the Bp reference strain K96243 was acquired and also purified. Moreover, because lipid A is the specific component of LPS that activates TLR4, lipid A was isolated from BP-1. HEK293 cells transiently transfected with murine or human TLR2, TLR2/1, TR2/6, or TLR4, and co-receptors CD14 and MD-2 were stimulated with these ligands (Figure 2A &2B). In both murine- and human-transfected cells, robust TLR4-dependent NF-κB activation was identified in response to LPS from both Bp strains and in response to BP-1 lipid A without evidence of TLR2 stimulation by these ligands. Additionally, identical stimulations with Bp LPS and BP-1 lipid A of cells transfected with human TLR4 and CD14 in the absence of MD-2 were performed, and no TLR4-mediated NF-κB activation was observed (Figure 2C).Figure 2B. pseudomallei LPS signals via TLR4. HEK293 cells were transiently transfected with (A) murine or (B) human TLR2, TLR2/1, TLR2/6, or TLR4; co-receptors CD14 and MD-2; and NF-κB-dependent firefly ELAM luciferase and control β-actin-dependent Renilla luciferase. In (C) HEK293 cells were transiently transfected with human TLR4 and co-receptor CD14 with or without MD-2; and firefly ELAM and Renilla luciferases. Cells were stimulated with media alone, (A & B) Pam3CSK4 1000 ng/mL, E. coli 0111:B4 LPS 10 ng/mL, BP-1 LPS 10 ng/mL, K96243 LPS 10 ng/mL, or BP-1 lipid A 10 ng/mL. NF-κB activation was measured by light emission (relative light units). In (A&B) data plotted are means ± standard deviations of duplicate or triplicate conditions. In (C) the means ± standard deviations of triplicate human TLR4-mediated relative light units (normalized to mean empty vector values) are plotted from parallel experiments with or without co-transfection of MD-2. For (A & B) § indicates p < 0.001 compared with empty vector stimulated with the same ligand, and † indicates p < 0.001 compared with TLR2 stimulated with the same ligand using analysis of variance followed by the Bonferroni post-test. Other comparisons are not shown for clarity. Each graph represents one of two independently performed experiments.These observations in a transfected cell line prompted us to examine the cytokine response to Bp and Bp LPS in primary cells. Therefore, bone marrow-derived macrophages from wild type, TLR2-/-, TLR4-/-, and TLR2/4-/- mice were stimulated with heat-killed BP-1, BP-1 LPS, K96243 LPS, or BP-1 lipid A. Pro-inflammatory cytokine TNF-α was quantified in cell supernatants by ELISA after 24 hours of incubation (Figure 3A). Compared to wild type cells, TNF-α production by TLR4-/- cells in response to BP-1 stimulation was significantly reduced, but was completely eliminated in response to BP-1 LPS, K96243 LPS, or BP-1 lipid A. In TLR2-/- cells, however, TNF-α production after stimulation with BP-1 was either the same as or greater than that produced by wild type cells in several different experiments (3.8 fold greater in the experiment shown). TLR2-/- cells invariably produced more TNF-α than wild type cells after stimulation with BP-1 LPS, K96243 LPS, or BP-1 lipid A (1.4, 2.0, or 2.2 fold greater, respectively, in the experiment shown). Furthermore, TLR2/4-/- cells demonstrated only partial reduction in TNF-α production compared to TLR4-/- cells stimulated with BP-1. Chemokine MIP-2 was also measured in the supernatants of wild type, TLR2-/-, TLR4-/-, and TLR2/4-/- cells stimulated with the same ligands, and a similar pattern to TNF-α production was observed (data not shown). A higher concentration of Bp LPS than control Escherichia coli LPS was used in these experiments because in a separate dose response experiment stimulating wild type macrophages, both strains of Bp LPS yielded several fold less TNF-α production than similar concentrations of E. coli LPS (data not shown).Figure 3Absence of TLR4, but not TLR2, impairs the macrophage cytokine response to B. pseudomallei. Bone marrow harvested from wild type, TLR2-/-, TLR4-/-, or TLR2/4-/- mice was cultured in the presence of L929 cell conditioned media for 5–10 days to promote differentiation of macrophages before plating and stimulating with media alone, Pam3CSK4 1000 ng/mL, E. coli 0111:B4 LPS 100 ng/mL, heat-killed BP-1 at a bacteria to cell ratio of 100, BP-1 LPS 1000 ng/mL, K96243 LPS 1000 ng/mL, or BP-1 lipid A 1000 ng/mL. Supernatants were harvested after 24 hours and (A) TNF-α or (B) IL-10 production was measured by ELISA. Data plotted are means ± standard deviations of quadruplicate samples. * indicates p < 0.05 and § indicates p = 0.001 compared with wild type cells stimulated with the same ligand, using analysis of variance followed by the Bonferroni post-test. Other comparisons are not shown for clarity. The TNF-α data displayed are from one of six independently performed experiments stimulating various combinations of wild type, TLR2-/-, TLR4-/-, or TLR2/4-/- macrophages with these ligands, and measuring TNF-α. In one of three experiments comparing cytokine responses from TLR2-/- macrophages to wild type macrophages, production of TNF-α in response to heat-killed Bp was not significantly increased. The IL-10 data displayed are from one of five independently performed experiments stimulating various combinations of wild type, TLR2-/-, TLR4-/-, or TLR2/4-/- macrophages with these ligands, and measuring IL-10. In two of three experiments comparing cytokine responses from TLR2-/- macrophages to wild type macrophages, production of IL-10 in response to heat-killed Bp was not significantly increased.The findings indicating augmented pro-inflammatory cytokine production in the absence of TLR2 when cells were stimulated with Bp, Bp LPS, and Bp lipid A led us to evaluate whether this was an IL-10-mediated phenomenon. IL-10 is an anti-inflammatory cytokine that may be released by cells upon TLR2 stimulation [18]. We therefore measured IL-10 in cell supernatants stimulated with heat-killed BP-1, BP-1 LPS, K96243 LPS, or BP-1 lipid A (Figure 3B). However, we did not identify any impairment in IL-10 production by TLR2-/- cells in response to these ligands. In general, the overall pattern of IL-10 production was again similar to TNF-α production, with augmentation of BP-1 LPS-, K96243 LPS-, or BP-1 lipid A-induced IL-10 release in the absence of TLR2.As the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) mediates both TLR2 and TLR4 signaling, MyD88-/- bone marrow-derived macrophages were therefore stimulated with heat-killed BP-1 and cytokine production assayed after 24 hours (Figure 4). Compared to wild type cells, TNF-α production by MyD88-/- macrophages was markedly curtailed.Figure 4MyD88 regulates TNF-α production by macrophages stimulated with B. pseudomallei. Bone marrow harvested from wild type or MyD88-/- mice was cultured in the presence of L929 cell conditioned media for 5–10 days to promote differentiation of macrophages before plating and stimulating with media alone or heat-killed BP-1 at a bacteria to cell ratio of 100. Supernatants were harvested after 24 hours and TNF-α production was measured by ELISA. Data plotted are means ± standard deviations of quadruplicate samples. § indicates p < 0.001 compared with wild type cells, using the t test. The data displayed represent one of two independently performed experiments.DiscussionIn this report, we demonstrate that heat-killed Bp activates TLR2 and TLR4. We further show that Bp LPS and Bp lipid A are TLR4 ligands and we identify augmented macrophage cytokine production induced by Bp, Bp LPS or Bp lipid A in the absence of TLR2.TLRs are innate immune receptors that recognize conserved pathogen motifs and initiate an inflammatory signaling cascade. We and others have demonstrated the essential roles of TLRs in host defense against a variety of pulmonary pathogens [5-9]. TLR2 is a promiscuous receptor that generally recognizes bacterial lipotechoic acid, peptidoglycans, or lipoproteins and functions as a heterodimer with either TLR1 or TLR6 [19-22]. LPS is the prototypical bacterial ligand for TLR4 [23] and is comprised of a core polysaccharide, polysaccharide side chains (the O-antigen) and lipid A (endotoxin), a glucosamine-based phospholipid. Lipid A usually interacts with TLR4 in addition to proteins CD14 and MD-2 to induce a pro-inflammatory signal [24]. MD-2 is a soluble molecule that is associated with the extracellular domain of TLR4 and is generally regarded as essential for LPS recognition by TLR4 [25-28]. Both TLR2 and TLR4 are therefore likely to initiate host defense signaling against Bp, a Gram-negative pathogen.As expected, our gain-of-function studies using murine and human constructs confirm a role for TLR2, in conjunction with TLR1 or TLR6, in initiating inflammatory signaling in response to heat-killed Bp. As TLR2 forms heterodimers with TLR1 or TLR6, the signal detected after transfection of TLR2 alone is likely explained by low level endogenous expression of TLR1 in HEK293 cells [22,29]. The role of TLR1 in facilitating TLR2-mediated NF-κB activation in response to Bp is further elucidated by our observation of minimal augmentation of the TLR2-dependent signal when we transfected a known hypo-responding variant human TLR1 plasmid [17].We also show that heat-killed Bp reliably signals via TLR4 and that Bp LPS isolated from two separate bacterial strains activates NF-κB in a uniquely TLR4-mediated fashion. We further demonstrate that Bp LPS subcomponent lipid A is a TLR4 ligand in cells transfected with either human or murine TLR constructs. Our observations that Bp LPS and lipid A are TLR4 ligands differ from the results of Wiersinga et al. [10], who used HEK293 cells stably transfected with TLR2/CD14 or TLR4/CD14 to show that Bp LPS signals through TLR2 but not TLR4. These differences may be explained by our co-transfection of MD-2, a molecule that is not endogenously expressed by HEK293 cells [29], but that we demonstrate is essential for TLR4-dependent signaling in response to Bp, Bp LPS, or Bp lipid A. While the LPS of a several bacteria signal via TLR2 rather than TLR4 [30-33], some LPS preparations that appear to stimulate TLR2 may be contaminated with lipopeptides [34]. Alternatively, there may be strain-specific differences in Bp LPS structure that influence recognition by TLR2 and TLR4. Although our results indicating roles for TLR2 and TLR4 in this gain-of-function system used heat-killed Bp, they corroborate recent work with live Bp by others [35].Our studies using primary murine cells provide additional evidence that Bp induces a TLR4-mediated inflammatory cytokine response and that Bp LPS and lipid A are TLR4 ligands. We demonstrate augmented Bp-induced TNF-α production in the absence of TLR2 alone and in the contemporaneous absence of TLR4. This differs from the TLR2-dependent TNF-α production by alveolar macrophages or whole blood stimulated with Bp by Wiersinga et al. [10] and may be attributable to the different cell types or bacterial strains used. However, our observations of enhanced cytokine production support their findings of reduced bacterial loads and improved survival in respiratory infection in TLR2-/- mice compared to wild type mice. We also observed consistent augmentation in cytokine production in TLR2-/- macrophages compared to wild type macrophages upon stimulation with Bp LPS or with ultrapure E. coli LPS, the control ligand. We have observed this phenomenon in previous studies of intracellular TNF-α production by primary bone marrow cells stimulated with Salmonella minnesota Re595 LPS, including in the absence of differential TLR7-dependent signaling between wild type and TLR2-/- cells [7]. Others have described a role for TLR2 agonists in suppressing TLR4-mediated inflammatory cytokine production [18] and a role for TLR4 in contributing to pulmonary inflammation in response to the TLR2 ligand lipoteichoic acid [36]. There are also other examples of cooperative TLR2 and TLR4 signaling in host defense [37,38]. However, we are not aware of specific descriptions of the absence of TLR2 influencing the inflammatory response to TLR4-specific stimulation alone. The mechanisms underlying this amplifying effect of TLR2 deficiency upon TLR4-mediated cytokine production by Bp LPS are unclear. If the presence of TLR2 is indeed suppressive, our data using LPS suggests that mere expression of the receptor rather than activation may be adequate to modify TLR4-specific signaling. Other reports indicate that TLR2 stimulation may induce production of the anti-inflammatory cytokine IL-10, promoting a Th2-type response [18,39-42]. However, our data do not support this mechanism of TLR2-mediated pro-inflammatory cytokine suppression. To the contrary, we show that Bp LPS-induced IL-10 production is augmented in the absence of TLR2, and, despite some differences largely attributable to experimental variability, generally mirrors the pattern of pro-inflammatory cytokine release observed. It may be that in the absence of TLR2, shared signaling molecules are more available to enhance activation of other TLRs, including TLR4. Further investigation will be required to elucidate the interaction of TLR2 and TLR4 in mediating recognition of Bp.Both TLR2 and TLR4 signal via the adaptor molecule MyD88, although TLR4 may also signal in a MyD88-independent fashion via adaptor molecule TIR-domain-containing adapter-inducing interferon-β (TRIF) [43]. Our observations that pro-inflammatory cytokine production by heat-killed Bp is largely abolished in the absence of MyD88 whereas TLR2/4-/- cells demonstrate only partial impairment in TNF-α release suggest that other MyD88-dependent receptors may be important in host recognition of Bp. Specifically, TLR5 and TLR9, receptors that recognize bacterial flagellin and CpG DNA, respectively, may account for some of this residual MyD88-dependent response. TRIF-mediated TLR4 signaling may also play a role in the innate immune response to Bp infection. These additional host defense pathways require further study. Importantly, our experiments have used only heat-killed Bp and we cannot discount differential signaling induced by live Bp. Thus, additional investigations with live bacteria are warranted.Our experiments also indicate that Bp LPS is a weaker inducer of TNF-α production in bone marrow-derived macrophages than E. coli LPS. Others have shown that Bp LPS is poorly pyrogenic but more mitogenic compared to Salmonella abortus equi [44]. Bp LPS may contribute to bacterial pathogenesis by modulating the host response and inhibiting macrophage killing [45]. Although Bp LPS is antigenically indistinguishable from the less virulent organism Burkholderia thailandensis [46], we have observed that Bp LPS may be a stronger inducer of pro-inflammatory cytokine production in macrophages than B. thailandensis LPS (unpublished data). Structural analysis of LPS preparations from these Burkholderia strains is required.ConclusionBp activates TLR2 and TLR4. In the presence of MD-2, Bp LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to Bp LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with Bp, Bp LPS or lipid A, cytokine production is augmented. Other MyD88-dependent signaling pathways may also be important in the host response to Bp infection. These findings provide new insights into critical mechanisms of host defense in melioidosis.AbbreviationsBp: Burkholderia pseudomallei; LPS: Lipopolysaccharide; MyD88: Myeloid differentiation factor 88; TLR: Toll-like receptor; TRIF: TIR-domain-containing adapter-inducing interferon-βCompeting interestsThe authors declare that they have no competing interests.Authors' contributionsTEW, RKE and SJS designed the experiments. RKE extracted LPS and lipid A. MJJ and TEW performed the experiments. TEW and SJS analyzed the data. TEW wrote the manuscript with input from MJJ, RKE and SJS.\n\nREFERENCES:\nNo References"
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batch_10/PMC2527564.json

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+{
+    "id": "PMC2527564",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527564\nAUTHORS: Rui Wang, Cheng Wu, Yanfang Zhao, Xiaoyan Yan, Xiuqiang Ma, Meijing Wu, Wenbin Liu, Zheng Gu, June Zhao, Jia He\n\nABSTRACT:\nBackgroundHealth related quality of life (HRQL) is a research topic that has attracted increasing interests around the world over the past two decades. The 36-item Short Form (SF-36) is a commonly used instrument for measuring HRQL. However, the information on Chinese adults' quality of life is limited. This paper reports on the feasibility of using the Mandarin version of SF-36 to evaluate HRQL in the population of Shanghai, China.MethodsA total of 1034 subjects were randomly sampled using a stratified multiple-stage sampling method in Shanghai. Demographic information was collected, and SF-36 was used to measure HRQL.ResultsInternal reliability coefficients were greater than 0.7 in six of the eight SF-36 dimensions, except social function and mental health. Intraclass correlation coefficients ranged from 0.689 to 0.972. Split-half reliability coefficients were higher than 0.9 in five SF-36 dimensions. Validity was assessed by factor analysis and correlation analysis. Our results were basically in accordance with the theoretical construction of SF-36. The average scores of most SF-36 dimensions were higher than 80. The primary influencing risk factors of HRQL included chronic diseases, age, frequency of activities, and geographical region, which were identified using multivariate stepwise regression.ConclusionOverall, HRQL in the population of Shanghai is quite good. The Mandarin version of SF-36 is a valid and reliable tool for assessing HRQL.\n\nBODY:\nBackgroundConception of health has been changed with the development of medicine and medical sciences since 1970's. Health is defined as a dynamic state of human wellbeing characterized by a physical, mental, and social potential which satisfies the demands of a life corresponding to age, culture, and personal responsibility, and not merely the absence of disease or infirmity. Health related quality of life (HRQL) is an individual's satisfaction or happiness with the dimensions of life insofar as they affect or are affected by \"health\" as defined above. HRQL has been introduced to assess people's health status. To date, a number of questionnaires have been developed to evaluate HRQL, and the 36-item Short Form Health Survey (SF-36) is the most commonly used one.SF-36 was developed from the Medical Outcomes Study or RAND Health Insurance Experiment [1]. It is a short-form derived from a larger 149-item instrument and is more precise than its predecessor, SF-20 [2]. SF-36 has been proven useful in monitoring population health, estimating the burdens of different diseases, monitoring outcome in clinical practice, and evaluating medical treatment effects. It has been translated into many languages with its content examined cross cultures [3-6]. In mainland China, the Mandarin SF-36 has been used in some surveys to assess the quality of life of the population with special chronic diseases [7,8]. However, the surveys on general populations were conducted only in Sichuan and Hangzhou [9,10].In this study, we aimed at (1) testing the reliability and validity of the Mandarin version of SF-36; (2) assessing health related quality of life in the population of Shanghai, China; and (3) evaluating risk factors that may significantly influence HRQL. The study was approved by the Second Military Medical University Ethics Committee.MethodsSampleShanghai is the biggest city in eastern China. It consists of 18 districts and 1 county that are geographically divided into 3 strata as urban, suburban, and rural regions. Using stratified multiple-stage sampling method, 4 residential areas in the urban region, 2 villages in the rural region, and 3 residential areas in the suburban region were selected following the sequence of district-block-residential area. A total of 1200 subjects older than 18 years of age were randomly sampled from those areas and 1034 subjects actually answered the questionnaires. The sample had 362 respondents from the Huangpu district, 336 from the Pudong district and 336 from the Songjiang district. The overall response rate was 86.17%. In order to analyze the reliability of the results, 10% of the total number of the respondents (i.e., 120 respondents) were randomly selected to take a retest by filling in the questionnaires again 2–7 days after the baseline test. At last 113 subjects took the retest. All respondents signed a written informed consent before participation.QuestionnaireThe questionnaire included general information and Mandarin version of SF-36 [See additional file 1: Questionnaire-bilingual]. General information was collected on age, sex, resident region, nationality, marital status, educational level, current job, family monthly income, height, weight, tobacco use, alcohol use, and frequency of activities. Body Mass Index (BMI) was calculated from height and weight. Since the current WHO BMI criterion is suitable for Caucasians rather than Asians, additional BMI categories for Asian populations are recommended by WHO [11]. Many Asian countries have also developed their own criterions, such as Japan [12]. We used the Chinese BMI criterion as follows: underweight was defined as BMI lower than 18.5 kg/m2, healthy weight as BMI from 18.5 to 23.9 kg/m2, overweight as BMI from 24 to 27.9 kg/m2, and obesity as BMI of 28 or more kg/m2[13]. In addition, respondents were asked whether they had been diagnosed by physicians with the following chronic conditions: hypertension, ischemic heart disease, cerebrovascular disorder, diabetes, chronic obstructive pulmonary disease (COPD), asthma, renal disorder, liver disorder, rheumatoid arthritis, osteoarthritis, anxiety, and depression, and at which age the disease had been first diagnosed. The use of medicine at the time of the interview was also recorded.The Mandarin version of SF-36 was translated from the IQOLA SF-36 Standard UK Version 1.0 by the experts of Zhejiang University, China. Its reliability and validity have been tested in the survey of Hangzhou, the capital of Zhejiang Province, southeast of Mainland China [10]. It was a brief self-administered questionnaire that generated assessment scores across eight dimensions of health: physical function (PF), role limitations due to physical problems (RP), bodily pain (BP), general health (GH), vitality (VT), social function (SF), role limitations due to emotional problems (RE), mental health (MH), and one single item dimension on health transition. The SF-36 dimensions can also be divided into two categories: Physical Component Summary (PCS) and Mental Component Summary (MCS), which represent the physical functioning and wellbeing, and emotional wellbeing, respectively.Field workThe survey was conducted from November 2005 to January 2006, using a self-finished interview method. Respondents filled in the questionnaires by themselves in their household or in local resident committees. The interviewers were social workers on the site who provided explanation without inducement on any unclear questions. Ten percent (10%) of the respondents had a repeatable accuracy check by filling in the questionnaires by themselves again a week later. The interviewers who interviewed the same respondent in the second time were different from the initial ones for the purpose of quality control. The performance of the interviewers was oversaw and coordinated by supervisors who examined questionnaires for any errors and ensured the quality of the survey. A valid questionnaire was the one that had been audited and signed by a supervisor. Both supervisors and interviewers were trained by the experts from Changhai Hospital and the epidemiologists from Second Military Medical University (SMMU).Data ManagementAll valid questionnaires were doubly input into the database by two independent professional data processors in the Department of Health Statistics of SMMU using software EpiData 3.1. Both manual checking and computer checking were conducted to find discrepancies.In the Pudong District, a total of 112 respondents' questionnaires were withdrawn from the statistical analysis due to one facilitator's failure to adhere to the study protocol. Three questionnaires from the Huangpu District were excluded because more than 80% items were missing. Therefore, after the data checking and validation, 919 effective questionnaires were used for data analyses in this study. Of the 113 respondents who agreed to be re-interviewed, 14 questionnaires were rejected because they were not completed in line with the study protocol, resulting in 99 questionnaires for the retest analysis.The missing values in the SF-36 dimensions were imputed as follows: if 50% or more items in one dimension were completed, the mean value of the completed items was used to impute the missing values. If more than 50% of the items were missing, the dimension score was excluded from the statistical analysis. In our survey, the item response rates were actually quite high. The average item response rate of the general information was 98.81%; the average response rate of the 36 items in SF-36 was 99.67%, ranging from 98.80% to 99.89%.Statistical analysisThe items and dimensions in SF-36 were constructed using the Likert method of summated ratings. The raw score of each of the eight SF-36 dimensions was derived by summing the item scores, and converted to a value for the dimension from 0 (worst possible health state measured by the questionnaire) to 100 (best possible health state). The raw score was then re-calculated across the dimension as follows:Transformed scale=[Actual raw score −lowest possible raw score Possible raw score range ]×100The PCS and MCS scores were calculated using the standard scoring algorithms [14-17].The SF-36 questionnaire was evaluated by reliability and validity. Split-half reliability was computed by correlating the scores of the odd half with those of the even half in each dimension of SF-36. Test-retest reliability was assessed by the differences between test and retest scores using a paired-sample t test. It was further assessed by intraclass correlation coefficient (ICC). A questionnaire with ICC value larger than 0.7 was usually considered satisfactory [18]. Internal consistency of the SF-36 items was assessed by Cronbach's α coefficient. A Cronbach's α value of 0.7 or higher was generally considered to be sufficient to demonstrate internal consistency [18]. Construction validity was assessed by correlation analysis and factor analysis using principal component analysis and quartimax rotation. Factor loadings larger than 0.50 within a particular dimension were considered to support its factor construction. The cumulative variance proportion was used to indicate the contributions of the factors [19].Statistical Analysis System (SAS) 9.1.3 and SPSS 10.0 were used for analyzing the survey data. Student t test, analysis of variance, and multivariate stepwise regression were applied to investigate the impact of various risk factors on quality of life.ResultsSample characteristicsA total of 919 subjects were utilized in the statistical analyses, including 509 female and 410 male. The age of all subjects ranged from 18 to 77 years with a mean age of 47 ± 13 years.Reliability1. Split-half Reliability AnalysisFive of the eight SF-36 dimensions (i.e., PF, RP, BP, VT, and RE) had the split-half reliability coefficient higher than 0.9, while the other three dimensions (i.e., GH, SF and MH) had the coefficient lower than 0.7. The lowest split-half reliability coefficient (0.368) was observed for the SF dimension (Table 1).Table 1Reliability and correlation of the SF-36 dimensions.ReliabilityCorrelationDimensionItem amountSplit-half reliabilityTest-retest difference meanICCCronbach's αCorrelations between dimensions and items insideCorrelations between dimensions and items outsidePCSMCSPF100.9090.153†0.9640.8620.257–0.9220.164–0.5640.7560.340RP40.974-1.020†0.7350.9500.869–0.9050.108–0.6550.6340.425BP20.904-1.224†0.8170.8630.837–0.9760.063–0.3630.6560.177GH50.5930.010†0.9520.8180.716–0.7850.123–0.6240.7560.517VT40.927-0.867*0.9720.7850.644–0.8680.155–0.6240.5330.690SF20.368-0.225†0.6890.3080.662–0.6870.067–0.3220.3260.508RE30.9670.000†0.8980.9510.877–0.9520.080–0.6140.3510.599MH50.647-0.204†0.8170.6910.596–0.8010.095–0.5790.2660.877* P-value = 0.034, † P-value > 0.052. Test-Retest Reliability AnalysisThe absolute mean differences between the test and retest scores ranged from 0.000 to 1.224. The paired-sample t test indicated that the difference between the test and retest scores was not statistically significant for seven of the eight dimensions, except the VT dimension (p-value < 0.05). The one-week ICC ranged from 0.689 (the SF dimension) to 0.972 (the VT dimension) for the eight SF-36 dimensions (Table 1).3. Cronbach's α AnalysisThe internal reliability of SF-36 was measured by Cronbach's α coefficient, which ranged from 0.308 (the SF dimension) to 0.951 (the RE dimension) for the eight SF-36 dimensions (Table 1).Validity1. Factor AnalysisThe results of the factor analysis were described in details in the previous study [20], and are briefly summarized here. Eight factors plus health transition item were created with a cumulative variance proportion of 71.25%. The RE and BP dimensions were perfectly in accordance with the theoretical construction of SF-36. Other items were basically correlated with the factors as expected.2. Correlation AnalysisSpearman correlation analysis showed that the correlations between the dimensions and items inside were higher than those between the dimensions and items outside. It was evident that the PF, RP, BP, and GH dimensions were correlated with PCS, while the VT, SF, RE, and MH dimensions were correlated with MCS. Among the eight SF-36 dimensions, PF was the best measure of physical health and MH was the best measure of mental health. In contrast, MH and BP were the poorest measures of the physical and mental components, respectively (Table 1).HRQLTable 2 showed the normative values of the SF-36 dimension scores by age and sex groups. The quality of life was reduced with increasing age. Female had lower scores than male in almost all subgroups, but in some subgroups female did report a better mental health. The SF-36 dimension scores were compared among different Chinese populations (Table 3): (1) every dimension score of the Shanghai population was higher than those of both Hangzhou population and American Chinese [10,21]; (2) the Shanghai population had higher scores than those of the Sichuan population in the RP, BP, SF, RE, and MH dimensions, but were similar to the Sichuan population in the PF, GH, and VT dimensions [9]; and (3) the SF-36 dimension scores of Hong Kong, Taiwan, American, and Canadian were lower than those of the Shanghai population in six of the eight dimensions, except GH and PF [22-25].Table 2Normative values of the SF-36 dimension scores by age/sex in the Shanghai population [Mean (SD)]AgeSexPFRPBPGHVTSFREMHPCSMCS18–29Female96.76(9.69)95.96(19.13)96.71(12.30)81.83(16.70)83.75(14.87)94.85(13.18)97.06(17.02)88.76(9.85)55.50(5.61)57.02(5.02)Male97.32(7.84)98.91(6.69)98.23(6.74)84.04(15.76)84.06(14.88)95.83(10.43)98.07(12.63)88.06(11.09)56.42(2.83)56.87(4.49)30–39Female96.15(6.78)96.54(16.46)94.45(14.13)72.00(18.13)75.92(13.26)93.95(11.99)96.92(17.40)85.05(12.48)54.53(3.76)55.13(5.07)Male96.85(7.77)97.83(14.74)99.09(4.46)77.78(16.02)76.74(16.47)95.92(9.15)100.00(0.00)84.17(14.99)55.82(3.14)55.36(5.44)40–49Female89.77(14.64)93.98(21.31)92.98(16.14)69.36(16.67)68.09(18.60)95.49(9.28)95.99(19.18)78.19(16.61)53.10(5.71)53.36(6.44)Male90.48(18.24)92.72(24.66)96.13(11.39)68.15(21.35)70.29(19.03)93.15(15.47)94.55(21.84)78.44(13.81)53.41(6.49)53.05(6.07)50–59Female87.62(13.35)91.44(26.23)94.56(13.21)64.79(18.49)68.61(18.16)94.09(11.68)93.12(24.41)80.89(14.50)51.91(5.54)53.88(6.67)Male88.44(15.48)93.20(23.84)94.90(14.32)64.87(17.59)69.20(18.03)94.60(12.53)94.13(22.82)79.71(16.23)52.41(5.48)53.67(6.66)60-Female79.14(18.20)90.96(27.49)90.00(17.24)58.18(19.41)65.60(19.42)92.77(12.37)91.97(25.28)80.58(15.05)49.00(6.67)54.22(6.76)Male83.18(13.63)92.80(23.93)91.58(17.04)61.76(17.46)68.94(16.77)92.99(12.23)95.96(17.06)81.88(14.43)50.19(5.80)54.97(6.22)TotalFemale89.05(14.56)93.16(23.53)93.75(14.67)67.89(19.21)70.97(18.38)94.26(11.58)94.55(21.83)81.85(14.64)52.51(5.92)54.41(6.33)Male90.55(15.01)94.50(21.30)95.70(12.49)69.88(19.66)72.78(18.23)94.33(12.66)95.85(18.84)81.64(14.75)53.36(5.62)54.45(6.11)Table 3The SF-36 dimension scores of different Chinese populations [Mean (SD)]DimensionShanghaiHangzhou10 Sichuan9 Hong Kong22 Taiwan23 American Chinese21 American24 Canadian25 (N = 919)(N = 1688)(N = 2249)(N = 2410)(N = 1191)(N = 156)(N = 2474)(N = 9423)Mean(SD)95%CIPF89.7(14.8)88.8, 90.782.2(19.8)90.6(15.4)91.8(12.9)92.6(11.5)79.4(23.4)84.2(23.3)85.8(20.0)RP93.8(22.6)92.3, 95.281.2(33.6)79.5(34.7)82.4(31.0)83.6(28.9)67.5(37.3)81.0(34.0)82.1(33.2)BP94.6(13.8)93.7, 95.581.5(20.5)85.6(18.4)84.0(21.9)82.4(16.8)62.3(21.9)75.2(23.7)75.6(23.0)GH68.8(19.4)67.5, 70.056.7(20.2)69.6(21.3)56.0(20.2)67.5(18.2)58.8(22.7)72.0(20.3)77.0(17.7)VT71.8(18.3)70.6, 73.052.0(20.9)70.3(17.1)60.3(18.7)65.3(15.2)59.0(20.3)60.9(21.0)65.8(18.0)SF94.3(12.1)93.5, 95.183.0(17.8)86.9(17.3)91.2(16.5)79.4(16.0)75.1(22.7)83.3(22.7)86.2(19.8)RE95.1(20.6)93.8, 96.584.4(32.4)76.5(38.5)71.7(38.4)71.3(37.0)61.2(43.7)81.3(33.0)84.0(31.7)MH81.8(14.7)80.8, 82.759.7(22.7)72.7(16.8)72.8(16.6)68.4(14.7)63.9(20.4)74.7(18.0)77.5(15.3)Risk factorsAnalysis of variance was first used to select the risk factors of HRQL. Region, gender, current job, age, current marital status, highest level of education, total income of family per month, frequency of activities, BMI, and chronic diseases were found influencing at least one dimension of SF-36. Multivariate stepwise regression was then applied using the SF-36 dimensions as the dependent variables and the risk factors mentioned above as the independent variables. The statistical significance level was set at 0.15 for both inclusion and exclusion of the independent variables in the stepwise process. The results indicated that the risk factors were different among the eight SF-36 dimensions (Table 4). Chronic diseases were evidently the most common risk factor reducing the scores of all SF-36 dimensions (p-value < 0.05). Its influence was relatively strong because its standardized regression coefficient was the largest one (in absolute value) for most of the SF-36 dimensions. Increasing age reduced quality of life in the PF, BP, GH, and VT dimensions, while manual work led to worse scores in the GH, VT and SF dimensions (p-value < 0.05). In contrast, high family income led to a good quality of life in the RE and MH dimensions. Similarly, frequent activities increased the quality of life in the PF, GH, VT, and MH dimensions (p-value < 0.05). The quality of life was improved with increasing education level in the RP dimension (p-value < 0.05). The unmarried had better quality of life in the VT and MH dimension (p-value < 0.05), but its standardized regression coefficients were quite low. The impact of gender and BMI was not statistically significant (p-value > 0.05). On the other hand, the impact of region was unclear: (1) living in the suburbs led to a better quality of life in the PF, GH, and VT dimensions, while the impact was negative in the SF, RE, and MH dimensions; and (2) living in rural regions resulted in a better quality of life in the RP, BP, and VT dimensions, but the impact was negative in the MH dimension with a high standardized regression coefficient.Table 4Standardized regression coefficients of the influence of risk factors on quality of life resulted from multivariate stepwise regression.FactorPFRPBPGHVTSFREMHRegion Urban Suburban0.16*-0.060.10*0.18*-0.08*-0.10*-0.08* Rural0.10*0.14*0.10*-0.25*Gender0.05Current job-0.08*-0.10*-0.07*Age-0.27*-0.08*-0.26*-0.15*Current marital status0.08*0.08*Highest level of education0.11*Total income of family/month0.050.07*0.15*Frequency of activities0.10*0.050.10*0.13*0.10*BMI0.05Chronic diseases-0.16*-0.21*-0.15*-0.23*-0.17*-0.16*-0.21*-0.08**P-value < 0.05Variable coding: Region (suburban = 1, other = 0; rual = 1, other = 0), gender (male = 1, female = 0), current job (manual worker = 1, office worker = 0), current martial status (unmarried = 1, married = 0), highest level of education (primary education and lower = 1, secondary/high education = 2, university education and higher = 3), family monthly income (less than 2000 Yuan = 1, 2000–4999 Yuan = 2, more than 5000 Yuan = 3), frequency of activities (never = 1, less than 4 times/month = 2, at least 1 time/week = 3, at least 1 time/day = 4), chronic disease (yes = 1, no = 0).DiscussionQuality of life is a study area that has attracted increasing interests over the past two decades. SF-36 has been used as an instrument for assessing quality of life world-wide. Normative data have also been obtained in many countries [25,26]. In mainland China, the Mandarin SF-36 has been used in a few surveys to assess the quality of life of general population and the population with special chronic diseases [7-10]. Our survey evaluated the feasibility of using the Mandarin SF-36 for investigating health related quality of life in the population of Shanghai, China.Statistical analyses used in this study included split-half reliability coefficient, ICC, paired-sample t test for the difference between the test and retest scores, and Cronbach's α. The results indicated that SF-36 was quite stable for the purposes of the study with a good internal consistency.In particular, the SF dimension had the lowest Cronbach's α coefficient in this study, which was consistent with other surveys using the Mandarin version of SF-36 [10,21,27,28]. The SF dimension also had the lowest ICC and split-half reliability, indicating there might be some problems in the conceptualization of social function. Traditionally, Chinese people don't think much about social function, and commonly say little or nothing about how the physical health or emotional problems would interfere with their social activities. In addition, the SF dimension included two questions as follows: (1) \"during the past 4 weeks, to what extent has your physical health or emotional problems interfered with your normal social activities with family, friends, neighbors, or groups?\", and (2) \"during the past 4 weeks, how much of the time has your physical health or emotional problems interfered with your social activities (like visiting with friends, relatives, etc.)?\" It appeared that the answers of the two questions had reverse orders, which may lead to the low reliability. The other reason may be the cultural diversity. In China, \"social activities\", translated as \"she hui huo dong\", refer to not only the everyday life within a family or one's circle of friends, but also the formal activities with other people such as going to a ballroom dancing event or attending a conference. The misunderstanding may result in the low reliability.MH and VT also had relatively low reliabilities [[10,21,27], and [28]]. The Cronbach's α coefficients for the VT and MH dimensions were 0.66 and 0.75 in the survey of Hangzhou [10], 0.72 and 0.71 in Sichuan [27], 0.74 and 0.77 in Hong Kong [28], 0.73 and 0.74 in American Chinese [21], and 0.78 and 0.69 in our study, respectively. This may be due to the characteristics of Chinese people since they are not used to talking about their feelings and emotions in public.Our results indicated a credible construction validity of SF-36 that was consistent with the outcomes of other surveys [10,29]. Factor analysis proved that our results were basically in accordance with the theoretical construction of SF-36. Correlation analysis indicated that each of the 36 items was highly correlated within the hypothesized dimensions, while relatively low correlations were observed between the items and other dimensions.Therefore, we concluded that SF-36 was acceptable and applicable for evaluating the quality of life in the general population of Shanghai, China. Compared our survey with other studies, American Chinese had the worst quality of life among different Chinese populations. Shanghai population had the best quality of life, even better than American and Canadian [9,10,21-25]. It should be noticed that the other studies in the comparison were undertook much earlier in time than our survey, and China has made impressive progresses in living standard during recent years. Especially, Shanghai is the financial and commercial center of China with the best medical and sanitation conditions. For example, the average life expectancy of the Shanghai population was 81.08 years old in 2007, which is slightly lower than the average life expectancy of Andorra, Macau, Japan, Singapore, San Marino, Hong Kong, and Canada. The infant mortality rate was 3.0‰ and maternal mortality rate was 6.68 deaths per 100,000 live births [30,31]. All these factors may lead to high HRQL in the Shanghai population.In addition, we found a very interesting fact about the normative values of the SF-36 dimension scores. Although the female had worse HRQL than the male in most subgroups, in some subgroups female did report a better mental health. The same results were found in other Chinese populations [9,10,21,22], a fact which is not usual in non-Chinese population. The outcome indicated the serious mental problems in Chinese men, which might be due to the huge stress in both work and life.We found that region, gender, current job, current marital status, the highest level of education, total income of family per month, frequency of activities, BMI, and chronic diseases had influences on at least one SF-36 dimension. But drinking and smoking did not significantly affect HRQL. When the interaction effects among these factors were excluded in multivariate regression, some risk factors such as resident region, chronic diseases, current job, frequency of activities, and age had strong influences on three or more SF-36 dimensions, while current marital status, the highest level of education, and total income of family per month affected only one or two of the SF-36 dimensions. These results were analogous to the previous study in Sichuan in which chronic condition, personal income, inhabitant places, age, and educational level were found to be the significant risk factors influencing quality of life, while marital status had impacts on few SF-36 dimensions [32]. All of the SF-36 dimensions were remarkably impaired by chronic diseases. People with chronic diseases had a worse quality of life than those without. It had been considered as the main risk factor impairing quality of life [7,8,32]. The PF, MH, GH, and VT dimensions were highly correlated with frequency of activities. Everyday activities, such as housework and walking, may help to stay healthy. The impact of age on quality of life was also notable. It was evident that the health problems became more and more serious with increasing age.There are some limitations in this study. Detailed information on non-responders were not collected, we were not sure whether there were differences between responders and non-responders. Although the interviewers received uniform training, there still might be influence of the interviewers' explanation on the results, and it was difficult to evaluate, which was also the limitation of this survey. Migrant workers, who make up a significant portion of the Shanghai population, were unable to be sampled because they remain officially registered in their place of origin. In addition, the sampling in the suburbs should be considered more carefully. Since the 1990s, urban population increased rapidly due to economic development and suburbanization in Shanghai. More and more people settled down in suburban regions, especially the group of white collars [33]. It made the distribution of suburban population more complicated.ConclusionIn summary, the Mandarin SF-36 is a valid and reliable questionnaire for evaluating both physical and mental health status. The quality of life in the Shanghai population is quite good compared with those in other Chinese populations. The primary influencing factors are region, chronic diseases, age, and frequency of activities. The key to improving quality of life includes the prevention and control of chronic diseases, and participating in moderate and regular activities. In addition, the elderly people should pay more attention on quality of life.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsRW assisted with the survey, completed the statistical analyses and drafted the manuscript. CW, YFZ, XYY and MJW assisted with the study, and XQM assisted with the survey and data analyses. WBL, ZG and JZ participated in the design of the study. JH conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:Supplementary MaterialAdditional file 1A health related quality of life survey in the population of Shanghai, China. English and Mandarin Chinese translations of a survey instrument for the HRQL study in China, including general information and SF-36 questionnaire.Click here for file\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2527611",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527611\nAUTHORS: Srdjan Denic, Nicolas Nagelkerke, Mukesh M Agarwal\n\nABSTRACT:\nBackgroundThe practice of consanguineous marriages is widespread in countries with endemic malaria. In these regions, consanguinity increases the prevalence of α+-thalassemia, which is protective against malaria. However, it also causes an excessive mortality amongst the offspring due to an increase in homozygosis of recessive lethal alleles. The aim of this study was to explore the overall effects of inbreeding on the fitness of a population infested with malaria.MethodsIn a stochastic computer model of population growth, the sizes of inbred and outbred populations were compared. The model has been previously validated producing results for inbred populations that have agreed with analytical predictions. Survival likelihoods for different α+-thalassemia genotypes were obtained from the odds of severe forms of disease from a field study. Survivals were further estimated for different values of mortality from malaria.ResultsInbreeding increases the frequency of α+-thalassemia allele and the loss of life due to homozygosis of recessive lethal alleles; both are proportional to the coefficient of inbreeding and the frequency of alleles in population. Inbreeding-mediated decrease in mortality from malaria (produced via enhanced α+-thalassemia frequency) mitigates inbreeding-related increases in fatality (produced via increased homozygosity of recessive lethals). When the death rate due to malaria is high, the net effect of inbreeding is a reduction in the overall mortality of the population.ConclusionConsanguineous marriages may increase the overall fitness of populations with endemic malaria.\n\nBODY:\nBackgroundMarriages between close biological relatives account for up to 60% of all marriages in many parts of Asia, Middle East and Africa [1]. A common finding among consanguineous populations is their long history of exposure to malaria. In fact, the frequency and degree of consanguineous marriages correlates with the geographic distribution and intensity of Plasmodium falciparum in the population [2]. Today, α+-thalassemia has become the most common monogenic disorder in humans potentially because it decreases the probability of death from infection with P. falciparum [3-5]. An earlier study has shown that the selection of many recessive alleles can be accelerated by inbreeding [6] and, recently, this has been demonstrated for α+-thalassemia in regions where malaria is endemic [7].The widespread practice of consanguineous marriages has conventionally been attributed to its multiple social benefits, e.g., the aggregation of economic wealth, the better treatment of spouse, and an increased family stability and security [1,8,9]. However, this theory of social benefits as being the main motivation for consanguineous marriages is unconvincing because the same benefits would also accrue in other populations, should they have chosen to be consanguineous. Moreover, consanguinity is found in societies within the same geographic area despite being racially, linguistically, religiously, and historically very heterogeneous [1,2]. It seems unlikely that such a cultural trait, which lowers population fitness, has spread just because of its socio-economic usefulness amongst these very diverse populations. In this study, we examined the potential positive effects of inbreeding (through selection of α+-thalassemia) versus its well established harmful consequences.MethodsThe modelThe genetic benefits (through α+-thalassemia allele) of inbreeding against the biological costs (via recessive lethal alleles) were evaluated in a stochastic model. The model has been verified by producing the results predicted by analytical methods (detailed in [7]) and uses the odds of survival of different α+-thalassemia genotypes from a field study [3]. In brief, the consanguineous and non-consanguineous populations were allowed to grow; their size (relative fitness) and α+-thalassemia allele frequency were compared. An initially large population (n = 1000) comprising of αα/αα genotypes was randomly seeded with α+-thalassemia using -α/αα genotype, so that the initial allele frequency was 0.03. The model was restricted to exclusively \"large\" populations as the effect of inbreeding on the selection of recessive and codominant alleles is significantly less in smaller populations [6,7]. Additionally, when malaria emerged as an epidemic infection 4,000 to 10,000 years ago, the Agrarian revolution had already caused a population explosion, an epidemiological pre-requisite for the appearance of malaria as an epidemic infection [10,11]. In this model, the population grows with the mating of a randomly chosen pair of individuals with a predetermined mean number of offspring; child's genotype is assigned using Mendelian rules of inheritance. After the mean coefficient of inbreeding (F) was allocated to a population, the couple was made consanguineous with a probability that equals the mean coefficient of relatedness, R (R = 2F). Biological relatedness of the couple was tested for each of the two alleles and, if found to be absent, another unmarried individual from the population was chosen and tested; this was continued until a biologically related individual was found -when a new marriage was arranged [7]. As only the surviving offspring become members of the next generation, there is no overlap between generations. In human consanguineous populations, highest reported F is 0.045 but, in the simulated experiment, the range was extended up to 0.09 because historically higher rates of inbreeding are possible [12,13].Mortality from P. falciparum is highest in the first five years of life; it decreases with subsequent infections and, during a single epidemic, malaria can kill up to 50% of a susceptible population. Mortality rates of malaria differ between various geographic areas; in the same area they may vary over time [5]. In this simulation, mortality rate was per generation, which was made constant throughout time. The survival probabilities of α+-thalassemia genotypes were derived from the field study data obtained in a population from an area with endemic malaria. The odds ratio (OR) for the development of severe forms of the disease that precede death from malaria, is lower in heterozygotes (with a single alpha gene deletion, -α/αα genotype, OR = 0.66) and is further decreased in homozygotes (with two alpha gene deletions, -α/-α genotype, OR = 0.40) than in those without any α-thalassemia mutation (αα/αα genotype, OR = 1.0) [3]. The survival of each of the three genotypes depends on the mortality from malaria and is shown in Figure 1. Survival (S) for the three genotypes is derived from the odds of death from malaria (r), which are extrapolated to equal the odds of the severe form of the disease, and mortality rate from malaria (p) so thatFigure 1The relative excess of α+-thalassemia allele frequency (red bars) and population size (blue bars) per generation in an inbred population when compared with a non-inbred population. The survivals (S) of the three genotypes used in this simulation are shown in the lower part of graph at the intersections with vertical lines which point to the results in the upper part of graph. The ratio of differential survival of genotypes = (S-α/-α - S-α/αα)/(S-α/αα - Sαα/αα).S = 1 - p'p'=rp(1−p+rp)In order to account for other causes of death, this result is scaled down to 0.7, i.e., 0.3 of all deaths are arbitrarily ascribed to non-malarial causes. The offspring of consanguineous families have a higher number of deaths (in the years prior to their reproduction) than offspring of non-consanguineous families; these deaths are due to homozygosity of harmful recessive alleles (inbreeding depression). An individual has on average of 1.4 recessive lethal alleles and the probability of excessive deaths due to inbreeding equals 0.7 F [13]. To account for this mortality in our model, all surviving children were exposed to an additional risk of 0.7F of dying before being allowed to reproduce; in the model, this consistently depressed population size in every generation by 0.7F.In each set of simulations, the starting allele frequency is 0.03, n = 1000. The mean number of children per couple varied between 5 and 14 in order to allow a positive population growth and prevent extinction of population when malaria mortality is high. However, all comparisons of inbred and outbred populations were performed using the same set of parameters except the one which was being tested. All results are the means of 300 simulation runs.Calculation of relative fitness and allele frequencyThe relative fitness (w) and allele frequency change (Δsp) in Figure 2 were determined fromFigure 2Effect of α+- thalassemia frequency on the relative fitness of inbreeding populations in the stochastic (upper panel) and analytic model (lower panel). In the upper panel, initial negative excess of relative fitness (blue bars) in the inbred population is the effect of recessive lethal alleles. An excess of α+-thalassemia allele (red bars) is seen after 5–6 generations – after allele frequency (black S-shape line) is increased to around 0.2. Relative excess of α+-thalassemia is maximal when its frequency is in the middle of the range (~0.35–0.7). Results are for n = 1000 and the ratio of differential survival = 1.39. In the lower panel, the calculated relative fitness includes only the effect of α+-thalassemia. The fitness ratio is the size of inbred population divided with that of outbred population. The results are for n → ∞ and the ratio of differential survival = 1.39.w=1−(1−F)(2pqsh+q2s)−FqsΔsp=(1−F)pqs[ph+q(1−h)]w+Fpqswh and s being 0.666 and 0.4, respectively, and corresponding to the ratio of differential survival of 1.39 and n→∞ in the stochastic model [7,14].Results and DiscussionWhen the mortality from malaria is low, consanguinity depresses the population with α+-thalassemia by causing an excessive number of deaths via recessive lethal alleles and by negligibly retarding the selection of α+-thalassemia allele (Figure 1a). The latter occurs when the difference between survival of -α/-α homozygote and -α/αα heterozygote genotypes is smaller than the difference between survival of -α/αα heterozygote and αα/αα homozygote (ratio of differential survival of genotypes < 1.0). This is also confirmed analytically, so, when F > 0 andS-α/-α - S-α/αα <S-α/αα - Sαα/αα,the sum of all the three products of genotype frequencies [q2(1 - F) + qF, 2pq(1 - F) and p2(1 - F) + pF)] and their survival is always smaller than when F = 0 [14]; this also applies to all allele frequencies (p and q = 1 - p). With an increase in mortality due to malaria, the ratio of the differential survival increases to 1.0 (Figure 1, lower graph) at which point, inbreeding has neither a negative nor a positive effect on the speed of selection of α+-thalassemia- the inbreeding depression being solely due to the effect of lethal recessive alleles. When the ratio of differential survival of genotypes becomes > 1.0, inbreeding starts to accelerate the selection of α+-thalassemia. This causes an excess of α+-thalassemia frequency in the inbred population, which increases its relative fitness in comparison to an outbred population (Figure 1b and 1c). This gain in relative fitness partially or fully compensates inbreeding depression (halting the expansion of inbreeding depression) due to recessive lethal alleles as clearly illustrated by Figure1b.As the death rate due to malaria increases, the relative excess of the frequency of α+-thalassemia in inbred populations increases further, and inbreeding depression may switch to inbreeding elevation, which takes place in populations with an F of 0.01 to 0.06 (Figure 1c), conspicuously within the range of F in consanguineous human populations (0 <F ≤ 0.045) reported over the last half century [13,14].When the initial frequency of α+-thalassemia is low (0.03), the onset of inbreeding elevation is delayed until the selection increases the frequency of α+-thalassemia (Figure 1b and 1c). The relative gain in fitness is greatest when the frequency of α+-thalassemia is around 0.5 (Figure 2); then the inbreeding elevation is observed immediately, from the first generation, and continuously thereafter in all populations with 0 <F < 0.05 (Figure 3). In native societies of the Arabian Peninsula, for example, α+-thalassemia allele frequency is around 0.3 and their mean F varies between 0.02 and 0.03, which suggests that overall consanguineous marriages have been genetically beneficial (before the control of malaria during the recent times) [15,16].Figure 3Inbred/outbred fitness ratios (FR) of populations with a high α+-thalassemia frequency over ten generations. At the start of simulation, the frequency of α+-thalassemia is 0.5. The black lines are trends for the first, fifth and tenth generation. FR > 1 is seen in every generation for all 0 <F < 0.05. This graph demonstrates robustness of inbreeding elevation found at all levels of inbreeding which were observed in human field studies, i.e., 0 <F ≤ 0.045 [13,14].The reasons for the practice of consanguineous marriages to begin and persist in a population (with a low frequency of α+-thalassemia) after it experiences an inbreeding depression (Figure 1, upper graph) remain unclear. It is also uncertain why consanguinity should increase in populations at the time of increased mortality, e.g., cholera and famine epidemics [17,18]. Figure 4 shows how the benefits of cousin-cousin marriages are obvious but not its hazards. When there are a very few α+-thalassemia alleles in any population, a carrier family may opt for a cousin-cousin marriage because of a relative shortage of suitable marriage partners – caused by the high mortality due to malaria. In such consanguineous unions, both spouses are likely to be α+-thalassemia heterozygotes. Conversely, if another member of the same family (who is likely to be a carrier), marries an outsider- the spouse is likely to be a non-carrier. On an average, 20% more children from consanguineous unions would survive malaria (Figure 4) – a survival advantage sufficiently big to be noticed by members of a population under stress. Hence, more consanguineous marriages would be encouraged to take place among α+-thalassemia-carrier families. A support for unequal benefit to families from consanguineous marriages (illustrated in Fig. 4) is the observation that children of consanguineous parents are themselves more likely to enter into consanguineous unions than the children of non-consanguineous parents [19]. However, even in non-carrier families, consanguinity may not be discouraged despite its genetic dangers (like childhood deaths and increased congenital malformations). This is because for any specific morbidity to be noticed, the difference from the reference (non-consanguineous, in this case) has to be sufficiently higher than 5%, but is generally much below this threshold [1,13,20,21]. In addition, the excess deaths due to recessive lethal alleles may be masked by a high overall death rate. This mechanism for initiation of consanguinity and its persistence suggests that consanguinity can originate de novo rather than spread by acculturation. Further, the genetic benefits from inbreeding may strengthen the development of endogamy and tribalism. This to some extent corroborates findings from India which has over 50,000 brotherhoods and the frequency of α+-thalassemia is higher in tribal than in city populations [4,22]. As the frequency of α+-thalassemia increases over time, more families would experience the net genetic benefits of consanguinity. This could explain the social permissiveness towards consanguinity in regions where it is beneficial, e.g., in the societies from regions with endemic malaria [23].Figure 4Initiation of consanguineous marriages. In family with α+-thalassemia, shortage of marriageable candidates leads to cousin-cousin and \"cousin\"-outsider unions. With the likely distribution of α+-thalassemia alleles shown above, on an average, 20% more children in this consanguineous union will survive malaria than from a non-consanguineous union. Here, the excess deaths due to inbreeding = 0.7F, i.e., 4.4% [13].The results presented agree with the historic conditions, which existed in the early human settlements, after the Agrarian revolution. At that time, populations increased rapidly due to better availability of food through farming and animal herding. However, the crowding, poor hygiene and proximity to animals contributed to the potential emergence of malaria and other epidemic infections [10,11]. Thus, when human survival became adversely affected by malaria, intra-family unions resulted in better survival of the offspring. A recent report of inbred families having more children than less inbred families in populations that never experienced malaria [24], further supports a role of human inbreeding as a facilitator of adaptation. In our globalized world with greater than ever mixing of populations, diseases like tuberculosis and AIDS are still the leading causes of death; protection against both is provided by codominant and recessive alleles [25,26] whose selection could be accelerated by inbreeding.ConclusionHuman inbreeding enhances the speed of fixation of recessive and codominant alleles. Consequently, the elimination of recessive lethal alleles is increased by an excessive mortality of children in consanguineous populations. However, an enhanced speed of selection of the codominant α+-thalassemia allele (in such inbred populations) increases the relative fitness against malaria. When mortality from malaria is high, this increase in fitness could offset the loss of life resulting from inbreeding. Therefore, consanguinity augments the fitness of a population with endemic malaria through its effect on α+-thalassemia allele.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsSD designed and obtained funds for the study, conducted the experiment, analyzed results and drafted the manuscript. NN analyzed results, provided analytical conformation of experimental results, and revised intellectual content of manuscript. MMA revised intellectual content and redrafted the manuscript. All authors contributed to writing and read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

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+{
+    "id": "PMC2527801",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527801\nAUTHORS: J Luo, K L Margolis, H-O Adami, A LaCroix, W Ye\n\nABSTRACT:\nA total of 138 503 women in the Women's Health Initiative in the United States were followed (for an average of 7.7 years) through 12 September 2005 to examine obesity, especially central obesity in relation to pancreatic cancer (n=251). Women in the highest quintile of waist-to-hip ratio had 70% (95% confidence interval 10–160%) excess risk of pancreatic cancer compared with women in the lowest quintile.\n\nBODY:\nPancreatic cancer ranks as the fourth leading cause of cancer death in the United States (Ekbom and Hunter, 2007). Besides tobacco smoking and chronic pancreatitis (Lowenfels et al, 1999), little is known of its aetiology, but recently, increasing evidence has suggested that obesity is a risk factor (Berrington de Gonzalez et al, 2003; Larsson et al, 2007). However, in most studies, the association seems to be weaker in women – who tend to gain weight more peripherally – than in men, suggesting that central adiposity may be a stronger risk factor for pancreatic cancer than body mass index (BMI). Few studies have investigated this aspect and the findings are inconsistent (Larsson et al, 2005; Sinner et al, 2005; Ansary-Moghaddam et al, 2006; Berrington de Gonzalez et al, 2006).We have used the large prospective Women's Health Initiative (WHI) study, with measured anthropometric factors, including hip and waist circumference, to examine the relationship between BMI, central adiposity, and pancreatic cancer risk.Materials and methodsWomen's Health InitiativeThe WHI, an ongoing, ethnically and geographically diverse, multi-centre clinical trial (CT) and observational study (OS), was designed to address some of the major causes of morbidity and mortality in postmenopausal women. Briefly, a total of 161 808 women aged 50–79 years were recruited at 40 clinical centres throughout the United States from 1 September 1993 through 1998. The WHI includes three overlapping CT components (hormone trial, dietary modification trial, and calcium/vitamin D supplementation trial) and an OS. All participants in WHI gave informed consent and were followed prospectively. Details of the scientific rationale, eligibility requirements and baseline characteristics of the participants in the WHI have been published elsewhere (Hays et al, 2003; Jackson et al, 2003; Langer et al, 2003; Ritenbaugh et al, 2003; Stefanick et al, 2003).The following participants were excluded from the original cohort of 161 808 : 14 849 with a history of cancer (except non-melanoma skin cancer) at baseline, 668 with no follow-up, 7491 with missing values of main exposures and confounders (including weight, height, waist circumference, hip circumference, smoking, and diabetes), and 297 (0.18%) with waist-to-hip ratio (WHR) values of beyond 4 s.d. (WHR<0.4 or WHR>1.2), leaving 138 503 women for analysis.All exposures in our analyses were collected at baseline for all participants. During the baseline clinical visit, trained and certified staff performed anthropometric measurements, including height, weight, hip and waist circumferences, and blood pressure. Body mass index was calculated as weight in kilograms divided by the square of height in metres. Waist circumference at the natural waist or narrowest part of the torso and hip circumference at the maximal circumference were measured to the nearest 0.1 cm. WHR was computed as the ratio of these two measurements. Weight changes during the participant's adult lives were obtained by self-report questionnaire, categorized as weight stayed stable (within 10 lb), steady gain in weight, lost weight as an adult and kept it off, and weight has gone up and down again by more than 10 lb.Information on demographic characteristics, medical history, and personal habits (lifestyle) were obtained by interview or by self-report using standardized questionnaires including age at enrolment (<55, 55–59, 60–64, 65–69, 70–74, 75–), smoking status (never, past, current) with information on how many years since quitting for past smokers and how many cigarettes smoked per day for ever smokers, physical activity and history of hypertension and diabetes while not pregnant.Women in the CT were followed through regularly scheduled examinations to ensure timely ascertainment of updated medical histories. All women in the CT were expected to attend annual clinical visits, with intermediate 6-month mail, phone or clinical contacts. The OS participants were contacted annually by mailed self-administered questionnaires. The completion rate of OS annual questionnaires was 93–96%. In this analysis, all participants were followed up until 12 September 2005. Initial reports of cancer were ascertained by self-administered questionnaires, and all self-reports of pancreatic cancer were confirmed by review of medical records, including pathology reports (if a biopsy or resection was done).Statistical analysesThe hazard ratio (HR) for pancreatic cancer was estimated using the Cox proportional hazards model. Different study cohorts (participation in OS or CTs, and different treatment assignments for all three CTs) were treated as strata in the model. In the multivariable models, we adjusted for age, smoking status (never, former smoking (quitted time⩾30 years, 20–29, 10–19, and <10 years), and current smoking (<4, 5–14, 15–24, 25 and more cigarettes per day)). As diabetes could be in the causal pathway between obesity and risk, we performed analyses (both adjusted and unadjusted) for this factor.We treated anthropometric measures as categorical (in quintiles) variables in the regression models, except BMI which was categorized (<22, 22–<25, 25–<30, 30–<35, and ⩾35 kg m−2). Tests for trend were performed by creating a continuous variable from the medians of the categories. In addition, to eliminate undiagnosed cases that might have experienced weight loss before completing the baseline questionnaires, we also performed analyses that excluded the first 2 years of follow-up. The proportional assumption was satisfied for all exposure variables of interest and potential confounding variables based on graphs of scaled Schoenfeld residuals (Hess, 1995).ResultsAs of 12 September 2005 with an average 7.7 years of follow-up, 251 incident cases of pancreatic cancer had been identified. Characteristics at baseline of selected variables by quintile of WHR are shown in Table 1. Compared with women with lower WHR, those with higher WHR were older, non-white, and less educated. Women with higher WHR were also more likely to be past or current smokers and tended to smoke more cigarettes per day and to have quit more recently, to have higher total energy intake, be less physically active, and have higher prevalence of diabetes and hypertension. WHR was positively associated with weight, BMI, waist and hip circumference.Among the tested anthropometric variables, only WHR was significantly associated with the risk of pancreatic cancer. After adjusting for potential confounders, women in the highest quintile of WHR had 70% (95% CI 10–160%) excess risk compared with women in the lowest quintile of WHR. When WHR was analyzed as a continuous variable, risk increased by 27% (95% CI 7–50%) per 0.1 increase. No association was observed between pancreatic cancer risk and other anthropometric variables, including height, BMI, hip circumference, waist circumference, and weight changes during adult life (Table 2).Finally, we repeated all the above analyses with the exclusion of the first 2 years of follow-up; findings remained broadly similar to those from the full analyses with RR=1.6 (95% CI: 1.0–2.6) comparing women in the highest to the lowest of WHR.DiscussionIn this large prospective study, we observed that central obesity measured by high WHR, rather than general obesity measured by high BMI, was associated with an increased risk of developing pancreatic cancer among postmenopausal women.Our result is consistent with most earlier studies for central adiposity and pancreatic cancer (Larsson et al, 2005; Patel et al, 2005; Ansary-Moghaddam et al, 2006; Berrington de Gonzalez et al, 2006), although not all (Sinner et al, 2005). In particular, the large prospective European prospective investigation into cancer and nutrition (EPIC) study (Berrington de Gonzalez et al, 2006) also observed significant increased risk associated with higher WHR (HR=1.24 (1.04–1.48) per 0.1 increase of WHR), but not with BMI. In fact, there was a weak or no association among women in all but 4 (Michaud et al, 2001; Calle et al, 2003; Pan et al, 2004; Patel et al, 2005) of the 16 studies published since 2000 on the association between the risk of pancreatic cancer and BMI that included women (Coughlin et al, 2000; Gapstur et al, 2000; Nilsen and Vatten, 2000; Hanley et al, 2001; Michaud et al, 2001; Wolk et al, 2001; Calle et al, 2003; Pan et al, 2004; Eberle et al, 2005; Fryzek et al, 2005; Larsson et al, 2005; Patel et al, 2005; Rapp et al, 2005; Sinner et al, 2005; Lin et al, 2007; Nothlings et al, 2007).The link between obesity and pancreatic cancer – similar to the plausible mechanism between diabetes and pancreatic cancer – may arise as a result of elevated fasting and postprandial glucose concentration, hyperinsulinemia, or both (Everhart and Wright, 1995; Gapstur et al, 2000; Batty et al, 2004; Jee et al, 2005). Many experimental studies (Pour and Stepan, 1984; Pour et al, 1990; Schneider et al, 2001; Wang et al, 2003; Hennig et al, 2004) and observational studies (Everhart and Wright, 1995; Gapstur et al, 2000; Huxley et al, 2005; Stolzenberg-Solomon et al, 2005) support the biological plausibility of higher insulin concentrations and insulin resistance in promoting pancreatic cancer development. If the induced insulin resistance is the underlying mechanism through which obesity increases the risk, then it is not surprising that we observed a stronger association with WHR abdominal adiposity than with BMI among postmenopausal women, because central adiposity is more strongly associated with glucose intolerance and increased insulin levels (Carey et al, 1997; Van Pelt et al, 2001; Sierra-Johnson et al, 2004; Tanko et al, 2004). In addition, the body fat distribution changes significantly following menopause, with a shift from preferential storage in gluteal/femoral regions to abdominal depots. Thus, our finding further suggests that the central adiposity is a better predictor of disease risk than BMI in postmenopausal women (Van Pelt et al, 2001).Strengths of our study include the prospective design, the large size of the cohort, the reasonably large number of cases, the high prevalence of obesity, including central adiposity, the detailed information on potential confounders, and the precise measurement of anthropometric factors. Measurement rather than self-reporting is particularly important for waist and hip circumferences, which are likely to be reported less accurately than height and weight. There is a possibility of misclassification among the cases, given the difficulty in diagnosis of this disease. However, this misclassification is likely to be non-differential with respect to anthropometric measurements, which may make our results conservative. A second possibility was weight loss because of undiagnosed disease, but results did not change after excluding the first 2 years of follow-up. It is unlikely that pancreatic cancer advanced enough to cause weight loss would remain undiagnosed for more than 2 years.In conclusion, our large prospective study shows that increased central adiposity was associated with an increased risk of developing pancreatic cancer among postmenopausal women.\n\nREFERENCES:\n1. Ansary-Moghaddam A, Huxley R, Barzi F, Lawes C, Ohkubo T, Fang X, Jee SH, Woodward M (2006) The effect of modifiable risk factors on pancreatic cancer mortality in populations of the Asia-Pacific region. Cancer Epidemiol Biomarkers Prev\n15: 2435–244017164367\n2. 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Lin Y, Kikuchi S, Tamakoshi A, Yagyu K, Obata Y, Inaba Y, Kurosawa M, Kawamura T, Motohashi Y, Ishibashi T, JACC Study Group (2007) Obesity, physical activity and the risk of pancreatic cancer in a large Japanese cohort. Int J Cancer\n120(12): 2665–267117304505\n24. Lowenfels AB, Maisonneuve P, Lankisch PG (1999) Chronic pancreatitis and other risk factors for pancreatic cancer. Gastroenterol Clin North Am\n28: 673–685, x10503143\n25. Michaud DS, Giovannucci E, Willett WC, Colditz GA, Stampfer MJ, Fuchs CS (2001) Physical activity, obesity, height, and the risk of pancreatic cancer. JAMA\n286: 921–92911509056\n26. Nilsen TI, Vatten LJ (2000) A prospective study of lifestyle factors and the risk of pancreatic cancer in Nord-Trondelag, Norway. Cancer Causes Control\n11: 645–65210977109\n27. Nothlings U, Wilkens LR, Murphy SP, Hankin JH, Henderson BE, Kolonel LN (2007) Body mass index and physical activity as risk factors for pancreatic cancer: the Multiethnic Cohort Study. Cancer Causes Control\n18: 165–17517219012\n28. Pan SY, Johnson KC, Ugnat AM, Wen SW, Mao Y (2004) Association of obesity and cancer risk in Canada. Am J Epidemiol\n159: 259–26814742286\n29. Patel AV, Rodriguez C, Bernstein L, Chao A, Thun MJ, Calle EE (2005) Obesity, recreational physical activity, and risk of pancreatic cancer in a large US cohort. Cancer Epidemiol Biomarkers Prev\n14: 459–46615734973\n30. Pour PM, Kazakoff K, Carlson K (1990) Inhibition of streptozotocin-induced islet cell tumors and N-nitrosobis(2-oxopropyl)amine-induced pancreatic exocrine tumors in Syrian hamsters by exogenous insulin. Cancer Res\n50: 1634–16392154330\n31. Pour PM, Stepan K (1984) Modification of pancreatic carcinogenesis in the hamster model. VIII. Inhibitory effect of exogenous insulin. J Natl Cancer Inst\n72: 1205–12086371345\n32. Rapp K, Schroeder J, Klenk J, Stoehr S, Ulmer H, Concin H, Diem G, Oberaigner W, Weiland SK (2005) Obesity and incidence of cancer: a large cohort study of over 145 000 adults in Austria. Br J Cancer\n93: 1062–106716234822\n33. Ritenbaugh C, Patterson RE, Chlebowski RT, Caan B, Fels-Tinker L, Howard B, Ockene J (2003) The Women's Health Initiative dietary modification trial: overview and baseline characteristics of participants. Ann Epidemiol\n13: S87–S9714575941\n34. Schneider MB, Matsuzaki H, Haorah J, Ulrich A, Standop J, Ding XZ, Adrian TE, Pour PM (2001) Prevention of pancreatic cancer induction in hamsters by metformin. Gastroenterology\n120: 1263–127011266389\n35. Sierra-Johnson J, Johnson BD, Bailey KR, Turner ST (2004) Relationships between insulin sensitivity and measures of body fat in asymptomatic men and women. Obes Res\n12: 2070–207715687409\n36. Sinner PJ, Schmitz KH, Anderson KE, Folsom AR (2005) Lack of association of physical activity and obesity with incident pancreatic cancer in elderly women. Cancer Epidemiol Biomarkers Prev\n14: 1571–157315941975\n37. Stefanick ML, Cochrane BB, Hsia J, Barad DH, Liu JH, Johnson SR (2003) The Women's Health Initiative postmenopausal hormone trials: overview and baseline characteristics of participants. Ann Epidemiol\n13: S78–S8614575940\n38. Stolzenberg-Solomon RZ, Graubard BI, Chari S, Limburg P, Taylor PR, Virtamo J, Albanes D (2005) Insulin, glucose, insulin resistance, and pancreatic cancer in male smokers. JAMA\n294: 2872–287816352795\n39. Tanko LB, Bruun JM, Alexandersen P, Bagger YZ, Richelsen B, Christiansen C, Larsen PJ (2004) Novel associations between bioavailable estradiol and adipokines in elderly women with different phenotypes of obesity: implications for atherogenesis. Circulation\n110: 2246–225215451790\n40. Van Pelt RE, Evans EM, Schechtman KB, Ehsani AA, Kohrt WM (2001) Waist circumference vs body mass index for prediction of disease risk in postmenopausal women. Int J Obes Relat Metab Disord\n25: 1183–118811477503\n41. Wang F, Herrington M, Larsson J, Permert J (2003) The relationship between diabetes and pancreatic cancer. Mol Cancer\n2: 412556242\n42. Wolk A, Gridley G, Svensson M, Nyren O, McLaughlin JK, Fraumeni JF, Adam HO (2001) A prospective study of obesity and cancer risk (Sweden). Cancer Causes Control\n12: 13–2111227921"
+}

batch_10/PMC2527802.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2527802",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527802\nAUTHORS: F Di Fiore, F Charbonnier, B Lefebure, M Laurent, F Le Pessot, P Michel, T Frebourg\n\nABSTRACT:\nNo Abstract\n\nBODY:\nSir,We reported last year, in the British Journal of Cancer, in a series of 59 metastatic colorectal cancer (MCRC) patients treated with cetuximab-based chemotherapy (CT), that KRAS mutation was highly predictive of treatment resistance and that progression-free survival was significantly increased in wild-type KRAS compared with mutant KRAS patients (Di Fiore et al, 2007). All the studies published so far have unambiguously confirmed that the presence of somatic KRAS mutation is indeed highly predictive of resistance to anti-EGFR antibodies in MCRC patients (Lièvre et al, 2006, 2008; Benvenuti et al, 2007; Frattini et al, 2007; Khambata-Ford et al, 2007; De Roock et al, 2008). Moreover, a large randomised controlled trial on panitumumab integrating KRAS genotyping has recently shown that, among 208 patients receiving panitumumab, 0 out of 84 mutants and 21 out of 124 (17%) wild-type patients were, respectively, responders (Amado et al, 2008). Therefore, KRAS genotyping should now be performed on a routine basis in patients with MCRC. In most of these studies, KRAS genotyping has been performed on primary colorectal tumours, whereas anti-EGFR antibodies are used to treat the metastatic disease. This strategy might, at least in certain circumstances, present two limitations. First, systematic KRAS genotyping in MCRC patients might be hampered in the future, at least for some patients, by the difficulty of obtaining tumour samples suitable for molecular analyses (and this might limit the use of anti-EGFR antibodies). Second, considering the genetic heterogeneity of colorectal cancers, the absence of detectable KRAS mutations in the primary tumour cannot formally exclude the presence of a KRAS mutation in metastases. For these two reasons, we think that detection of KRAS mutation in the blood of patients with MCRC may have a clinical interest in the context of anti-EGFR therapies and we would like to highlight in this letter the potential interest of such a strategy. Although several studies have shown the presence of mutant DNA in blood from patients with colorectal neoplasia, only positive results are informative. Therefore, one should consider the development of combined tests indicating in blood, first the presence of tumour DNA, then the status of KRAS. In MCRC, hypermethylated DNA can be used as a blood tumour molecular marker. For instance, hypermethylation of the RASSF2 gene has frequently been detected in colorectal adenoma and invasive carcinoma (Park et al, 2007), and we found, in a series of 32 patients with MCRC, that RASSF2 was hypermethylated in 79% of the tumours (unpublished results). In addition to RASSF2, other targets may be used to ensure a sensitive detection of tumour DNA, if RASSF2 is not found hypermethylated. For sensitive detection on a routine basis of KRAS mutation, several methods, shown to be more sensitive than conventional dye-labelled dideoxynucleotide sequencing, are now available, such as, SNaPshot or PCR-LCR assays (Di Fiore et al, 2007), or allele-specific real-time PCR (De Roock et al, 2008; Lièvre et al, 2008). We used this strategy of combined blood assays to analyse two patients who received cetuximab-based CT, one responder and the other showing a progressive disease after anti-EGFR therapy. We screened the plasma of these patients for the presence of methylated DNA, using a classical methyl-specific assay exploring the RASSF2A promoter after bisulphite treatment, and then for the presence of KRAS mutation using real-time PCR, performed in the presence of a peptide nucleic acid (PNA) sequence specific for the wild-type KRAS codons 12 and 13, which inhibits amplification from the wild-type template. The first patient, a 67-year-old man, received cetuximab and irinotecan regimens for a peri-hepatic lymph node tumour recurrence 12 months after surgery for liver metastases, and after 3 months, evaluation revealed disease progression. In patient plasma collected before the beginning of cetuximab CT, the combined assays revealed the presence of hypermethylated RASSF2 (Figure 1A) and the presence of mutant KRAS. Sequencing analysis of the PCR product obtained in the presence of the PNA revealed the same KRAS mutation (Figure 1B), as the one previously detected in the colorectal tumour and liver metastases. The second patient, a 76-year-old man, received, in second line, cetuximab plus irinotecan CT for hepatic metastasis occurring 4 years after curative surgery for a bifocal CRC adenocarcinoma, and this treatment allowed control of the disease with the duration of response of 10 months. In this patient, the combined assays performed on the plasma collected before cetuximab treatment showed the presence of hypermethylated RASFF2A (Figure 1) but the absence of mutant KRAS.We therefore suggest that, in the forthcoming clinical trials on anti-EGFR antibodies in MCRC, which integrate KRAS genotyping, it is probably useful to collect blood samples before treatment and that the clinical interest of such combined blood tests, using the presence of hypermethylated DNA, as tumour DNA marker, and a sensitive method for KRAS mutation detection, should be evaluated on large series of MCRC patients.\n\nREFERENCES:\n1. Amado GR, Wolf M, Peeters M, Van Cutsem E, Siena S, Freedman DJ, Juan T, Sikorski R, Suggs S, Radinsky S, Patterson SD, Chang DD (2008) Wild-Type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol\n26: 1626–163418316791\n2. Benvenuti S, Sartore-Bianchi A, Di Nicolantonio F, Zanon C, Moroni M, Veronese S, Siena S, Bardelli A (2007) Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies. Cancer Res\n67: 2643–264817363584\n3. De Roock W, Piessevaux H, De Schutter J, Janssens M, De Hertogh G, Personeni N, Biesmans B, Van Laethem JL, Peeters M, Humblet Y, Van Cutsem E, Tejpar S (2008) KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Ann Oncol\n19: 508–51517998284\n4. Di Fiore F, Blanchard F, Charbonnier F, Le Pessot F, Lamy A, Galais MP, Bastit L, Killian A, Sesboüé R, Tuech JJ, Queuniet AM, Paillot B, Sabourin JC, Michot F, Michel P, Frebourg T (2007) Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by cetuximab plus chemotherapy. Br J Cancer\n96: 1166–116917375050\n5. Frattini M, Saletti P, Romagnani E, Martin V, Molinari F, Ghisletta M, Camponovo A, Etienne LL, Cavalli F, Mazzucchelli L (2007) PTEN loss of expression predicts cetuximab efficacy in metastatic colorectal cancer patients. Br J Cancer\n97: 1139–114517940504\n6. Khambata-Ford S, Garrett CR, Meropol NJ, Basik M, Harbison CT, Wu S, Wong TW, Huang X, Takimoto CH, Godwin AK, Tan BR, Krishnamurthi SS, Burris III HA, Poplin EA, Hidalgo M, Baselga J, Clark EA, Mauro DJ (2007) Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J Clin Oncol\n25: 3230–323717664471\n7. Lièvre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, Ychou M, Bouché O, Landi B, Louvet C, André T, Bibeau F, Diebold MD, Rougier P, Ducreux M, Tomasic G, Penault-Llorca F, Laurent-Puig P (2008) KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol\n26: 374–37918202412\n8. Lièvre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF, Côté JF, Tomasic G, Penna C, Ducreux M, Rougier P, Penault-Llorca F, Laurent-Puig P (2006) KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res\n66: 3992–399516618717\n9. Park HW, Kang HC, Kim IJ, Jang SG, Kim K, Yoon HJ, Jeong SY, Park JG (2007) Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers. Int J Cancer\n120: 7–1217013898"
+}

batch_10/PMC2527814.json

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+{
+    "id": "PMC2527814",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527814\nAUTHORS: S Ciatto, N Houssami, F Martinelli, R Bonardi, F H Cafferty, S W Duffy\n\nABSTRACT:\nLittle is known about long-term outcomes following a second breast cancer diagnosis. We describe the epidemiology, characteristics and prognosis of second breast cancers in an Italian cohort. We identified women with two breast cancer diagnoses from 24 278 histology records at a Tuscan breast cancer service between 1980 and 2005, and determined their survival status. Disease-specific survival from second diagnosis was examined using Cox regression analyses. Second cancers were identified in 1044 women with a median age of 60 years. In all 455 were ipsilateral relapses and 589 were contralateral cancers. Median time between first and second diagnosis was 63.4 months. The majority of second cancers was small invasive or in situ tumours. Estimated 10-year survival from a second cancer diagnosis was 78%. Survival was poorest when the second cancer was large (HR=2.26) or node-positive (HR=3.43), when the time between the two diagnoses was <5 years (HR=1.45), or when the diagnosis was in an earlier epoch (HR=2.20). Second tumours were more likely to be large or node-positive if the first breast cancer had these features. Prognosis following a second breast cancer in this cohort was generally good. However, large or node-positive second tumours, and shorter intervals between diagnoses were indicators of poorer survival.\n\nBODY:\nBreast cancer (BC) is estimated to be the most prevalent cancer in the world (Parkin et al, 2005). Earlier diagnosis and better therapy have improved survival from BC. Information relevant to the care of this group of such women, who have an elevated risk of a second BC, is relevant to population health and clinical practice. Few studies have examined a second BC in well-defined series, and little is known about the characteristics and long term outcome in affected women (Grunfeld et al, 2002).We report on the largest clinically defined cohort (to date) of second BC events, based on consecutive cases in a major breast service in Tuscany, and including women with ipsilateral breast relapse (IBR) and contralateral breast cancer (CBC). We aim to describe the epidemiology and clinical characteristics of second BCs, and to identify factors associated with poorer survival following a second BC diagnosis. The focus of our study is therefore prognosis following second BC events and not predictors of its occurrence.Materials and methodsEligible participants consisted of all women with a prior primary BC (invasive or in situ) who were diagnosed with a second metachronous BC (⩾6 months after the first cancer) in 1980–2005 at the study centre. We searched the clinical and pathology archives of the Centro per lo Studio e la Prevenzione Oncologica (CSPO), identifying women with histology records (surgical or needle histology) indicating two BC diagnoses separated by at least 6 months. Medical records were then reviewed to verify eligibility. Centro per lo Studio e la Prevenzione Oncologica is Florence's main breast screening and diagnostic service, and is the only centre in the region that provides follow-up services for women with BC. Surveillance consists of two-view mammography complemented by clinical examination (with more frequent clinical examination in the initial 5 years in women treated with breast conservation). Centro per lo Studio e la Prevenzione Oncologica archives have ongoing data linkage to population cancer and mortality registries in the Tuscan region.We use the term second BC (‘second cancer’) when describing all cases, but define two subgroups eligible for inclusion: women with (1) ipsilateral breast relapse (IBR), who developed a second cancer in the breast that was previously affected (including those with breast relapse and concomitant axillary disease); and (2) women with CBC, who developed a second cancer in the opposite breast. Women presenting with metastatic cancer at diagnosis of the second BC, who represent a very small proportion of women, were not considered for this study as histology verification is inconsistently available in these cases; inclusion might bias estimates of survival from second cancer diagnosis; and owing to difficulty in ascertaining whether the metastases were related to the first or second cancer event. Women presenting primarily with nodal metastases or chest wall recurrences (following mastectomy for the first cancer) were not eligible for inclusion in this study.Data retrieved from clinical records included date of birth; date of diagnosis, histology and pathological T and N category of the first BC; date of diagnosis, side, histology and pathological T and N category of the second cancer; presence or absence of symptoms at diagnosis of second cancer; surgical management; and date of last follow-up. Survival status was assessed directly for patients regularly followed up at CSPO or according to the regional Mortality Registry for cases lost to active follow-up. Linkage with the Mortality Registry for cause of death was complete to 31 December 2005, and ascertainment of outcomes was 97% complete to 31 December 2006. Data on treatment were not available for this study, but in our setting, it comprises radiotherapy following breast conservation and (since about 1988) adjuvant systemic therapy.Statistical analysisAnalyses are presented for all second cancers collectively and for each group (IBR and CBC). Descriptive data are reported on key variables including histology and tumour stage distribution for both the first and the second cancers. Time between occurrence of the first cancer and histological diagnosis of a second cancer (‘disease-free interval’, DFI), and time from second diagnosis to BC death, were calculated. When comparing features of the second tumour with features of the first tumour, data are paired and the McNemar's χ2 test was used. When comparing the IBR cases with the CBC cases, groups are independent and the χ2 test was used.To determine the prognostic effect of features of the second cancer, we examined disease-specific survival from the second cancer diagnosis. Survival from diagnosis of the first cancer will be the subject of a separate study. For subjects who were alive at the end of follow-up or had died from a different cause, observations were censored at date of their last observation (normally 31 December 2006) or date of death. Ten-year survival was estimated using the Kaplan–Meier method. Multivariate Cox regression analysis was performed using the following covariates: (a) age at second cancer diagnosis (<50, 50–69, ⩾70 years); (b) time from first to second cancer (DFI); (c) stage (pT and pN category) of the second cancer; (d) time period in which the second cancer was diagnosed (1980–97, 1998–2005); and (e) type of second cancer (IBR or CBC). Models were fitted separately for IBR and CBC cases, as well as for all second cancers. Results are expressed in terms of hazard ratios (HR) and 95% confidence intervals (CI).The Cox regression model was used to identify features of the second tumour that had the most significant adverse influence on prognosis following its diagnosis. Multivariate logistic regression analyses were then used to identify which features of the first BC predicted such second BC. The covariates under consideration were (a) age at first cancer diagnosis (<50, 50–69, ⩾70 years); (b) stage of the first cancer (pT and pN category); and (c) histology of the first cancer (invasive ductal, DCIS, other invasive). Note that this analysis is designed to predict particular features of a second tumour and not the occurrence of a second tumour.All statistical analyses were performed using the STATA software, release 8.0.ResultsSecond BCs were identified in 1044 women from 24 278 breast histology records: IBR in 455 subjects and CBC in 589 (Table 1). The median age at diagnosis of the second cancer was 60 years (IQR 51–70). Follow-up was available in all but 36 cases (20 IBR, 16 CBC), which were excluded from outcome analyses but retained in descriptive analyses. The median follow-up period from the occurrence of the first cancer was 13.7 (IQR 9.0–18.1) years. There were 181 BC deaths and 62 from other causes (Table 1). Second cancers were more likely to be detected asymptomatically (67.0%) than symptomatically (33.0%, P<0.001). However, the proportion of symptomatic cases did not differ between IBR and CBC (P=0.21, Table 1).The majority of second cancers was small invasive or in situ (77% were pTis or pT1). DCI comprised 12%, invasive ductal 56% and other histological types 32%. Of CBC cases, 56% were node negative, 20% were node positive, and in 24% node status was unknown. There was a significant difference between IBR and CBC with respect to the histological type of the tumour (P<0.0001), more IBR cases being DCIS, whereas more CBC cases were invasive lobular. Similarly, there was a significant difference between the two groups in the histological types of the first tumour (P<0.0001); more IBR had been diagnosed with DCIS previously compared with those with CBC (17.1 vs 5.8%). Significant differences in the distribution of pT categories between IBR and CBC were also seen for both first and second tumours (Table 1).Table 2 summarises data on size (pT) of second tumours according to the size of the first tumour and the type of second cancer (IBR or CBC). For women diagnosed with CBC, the second tumour tended to be smaller than the first. Of those with known tumour size of both first and second cancers, 80.5% (409/508) of second cancers were pT1 or smaller, compared with 58.5% (297/508) of first cancers (P<0.0001). However, this difference was not observed in women with IBR, whose second tumour was more likely to be of a similar size to the first tumour (83.3% (334/401) of second cancers and 81.5% (327/401) of first cancers were pT1 or smaller, P=0.5).Figure 1 shows Kaplan–Meier survival curves for IBR, CBC and all second cancers. The estimated 10-year survival rate from IBR was 77.9%, from CBC, 77.3% and overall in second cancers 77.6%. In univariate analysis significantly poorer survival was observed with younger (<50 years) and older (>69 years) age at second cancer diagnosis (P=0.03), a short interval from first to second cancer (P=0.001), larger tumour size (P<0.0001), positive nodes (P<0.0001), and cancers diagnosed during the earlier time period (P<0.0001). These effects were similar for IBR and CBC cases and so both groups were included in the final multivariate model. The multivariate Cox regression model is presented in Table 3 showing the proportional hazard ratios (HR) and 95% CI for second BC variables. The strongest predictor variables for survival in this model were pT and pN categories, although time between first and second cancer, and epoch of diagnosis of second cancer, also remained significant. There was no evidence of a difference in survival between IBR and CBC.The Cox model indicates that second cancers that were 2 cm or larger or node positive had a poor prognosis. Table 4 summarises multivariate analysis of the features of the first cancer that predicted these features in the second tumour: women whose first cancer was large or node positive at diagnosis were more likely to have large or node-positive second cancer.DiscussionThe characteristics of second BCs in women presenting to a major centre in Tuscany represents the largest series to date to include both IBR and CBC, with data on outcomes in almost all subjects. As BC survival is improving, clinicians will be providing care to increasing number of women at risk of developing a second cancer in either breast. Women who experienced IBR or CBC in this study were on average around 60 years of age when they experienced the second BC event. This is not an old population in the context of life expectancy in developed countries. These cancers are more likely to be diagnosed in asymptomatic women, and are generally smaller tumours than the initial cancer in the same women.Overall 10-year survival from diagnosis of the second cancer was 77.6%, and the median time between first and second cancer diagnosis was 63.4 months. A shorter time from first till second cancer diagnosis was associated with poorer survival. Ten-year survival estimates in our study are similar to survival estimates of 75% observed for first invasive cancers in the Swedish two-county study (Tabar et al, 1992), and the 73% observed in first invasive cancers diagnosed in the 1990s in the West Midlands, UK (G Lawrence, personal communication).Many studies have looked at IBR and reported on factors predicting local recurrence. We have taken a different approach by focusing on the identification of features associated with prognosis once a second BC has occurred (rather than predictors of recurrence) and have included CBC as well as IBR. Most studies of CBC have included small number of cases and limited follow-up (Grunfeld et al, 2002). The exception to this is a study that reported on stage-related survival in CBCs only and showed good prognosis in early-stage tumours (Schootman et al, 2006). We recognise that IBR and CBC are essentially different events and for this reason we have reported data separately in the two groups. Our summary of key features (Table 1) highlights both similarities and differences in second cancers. Variables associated with prognosis in survival analysis were similar for both IBR and CBC. We were unable to report on tumour grade and hormone receptor status because of incomplete or non-availability of such data. It should be noted that Rack and colleagues, in a study of locoregional breast relapse found that tumour grade was not independently associated with risk of death following relapse (Rack et al, 2003).The distribution of times between first and second cancer diagnosis in this cohort (both IBR and CBC) provides evidence supporting long-term follow-up of women after BC. Currently, some groups recommend only limited follow-up (National Institute for Clinical Excellence, 2002; The Association of Breast Surgery @ BASO, 2005). However, our study suggests that a recommended follow-up of 10 years or even longer may be more appropriate (Smith et al, 1999; Khatcheressian et al, 2006). A recent study from Edinburgh, based on 108 relapses (including 35 cases of CBC) makes a similar point, and reports that treatable breast relapse occurred at a constant rate for at least 10 years (Montgomery et al, 2007). The benefits of extending surveillance must be weighed against potential disadvantages in terms of overdiagnosis and overtreatment although these effects are likely to be small for this cohort.Surgical management of CBC was more often breast conservation than mastectomy, in keeping with the preponderance of small cancers in this group. In addition, most women with CBC had node-negative disease, although data on nodal status were not known in about a quarter of cases (nodes either not excised or data not notified to the study centre). Although local excision of in-breast relapse is associated with lower local control than salvage mastectomy (van der Sangen et al, 2006), a 40% breast conservation rate in women with IBR in our study might reflect feasibility of local excision as 62% were small tumours.Our data do not represent complete population data; however, the study centre is the region's main breast diagnostic service with linkage to population registries, and is the only service in the Tuscan region which provides organised surveillance for women with a past BC history. We therefore consider that the majority of women in our case definition will have been identified and included. It is also relevant that our study concerned subjects with two BCs and does not allow calculation of predictors of the occurrence of a second cancer event nor rates of IBR and CBC. These were not within the scope of this evaluation; such data on these issues have been previously reported (Kurtz et al, 1988; Rack et al, 2003; Montgomery et al, 2007).One of the clear findings of this study is that the majority of second BCs was detected in asymptomatic women. These data suggest that ‘early detection’ of the second cancer may be occurring during routine follow-up of women with BC. A recent case–control study found mammography surveillance was associated with improved survival in older BC survivors (Lash et al, 2007). As we are considering survival from diagnosis of the second cancer, estimation of the effect of early detection (based on symptom status) is likely to be subject to lead-time and length biases. For this reason we have not included this variable in survival analysis for purposes of this study. However, we are currently collecting additional data on symptoms and mode of diagnosis of second cancers in our cohort to validly quantify the extent and potential impact of early detection in women with second cancer events.The high proportion of asymptomatic second cancers is consistent with finding that overall second cancers were smaller than first cancers, although clearly evident for only CBC. As CBC is essentially a new cancer event, screening may play a larger role in its diagnosis. Ipsilateral breast relapses are predominantly recurrences of the initial tumour and so the observed differences in tumour size may reflect the differing biological nature of these two cancer events.Histological types of tumours, for both the first and second cancer, also differed in distribution between IBR and CBC. This needs to be interpreted in the context of the long timeframe of the study (with possible variability in pathology reporting criteria and pathologists) and that some types were not specified. The interesting aspect of differences in the distribution of tumour histology is that it was largely determined (in second cancers) by a significantly higher proportion of DCIS in IBR than CBC, and a significantly higher proportion of invasive lobular cancer in CBC than IBR. Differences in distribution of histology of the first cancer were similarly influenced by a significantly higher proportion of DCIS in IBR than CBC.Poorer disease-specific survival was associated with shorter times from the first to second cancer event, diagnosis of the second cancer in an earlier time period, second cancer tumour size ⩾2 cm, and positive nodes. The association between poorer prognosis and short time from the initial cancer to in-breast relapse is well known, but such an association has not previously been clearly demonstrated for CBC (Ciatto et al, 2004). The association between earlier epoch of diagnosis and poorer survival has been reported by others in analyses of population data in both primary early and metastatic BC (Chen et al, 2007; Ernst et al, 2007; Dabakuyo et al, 2008), so we presume this is mainly indicative of therapy effect. It is also possible that some of this effect relates to early detection.Although all the prognostic variables we have described are relevant to clinicians providing care to women with a second BC, based on the strongest predictor variables for survival in our model, pT and pN categories are likely to be more clinically relevant in prognostication, although the occurrence of a second cancer soon after the first may be a warning sign of a more aggressive tumour and poorer outcome. The most powerful predictive features of the first BC, in terms of predicting second cancers with poor prognostic features (second cancer tumour size ⩾2 cm or positive nodes) were larger tumour size and positive nodes. A larger first BC (⩾2 cm) was a significant predictor of node metastases on diagnosis of the second cancer.\n\nREFERENCES:\n1. Chen SL, Hoehne FM, Giuliano AE (2007) The prognostic significance of micrometastases in breast cancer: a SEER population-based analysis. Ann Surg Oncol\n14: 3378–338417899293\n2. Ciatto S, Miccinesi G, Zappa M (2004) Prognostic impact of the early detection of metachronous contralateral breast cancer. Eur J Cancer\n40: 1496–150115196532\n3. Dabakuyo TS, Bonnetain F, Roignot P, Poillot ML, Chaplain G, Altwegg T, Hedelin G, Arveux P (2008) Population-based study of breast cancer survival in Cote d'Or (France): prognostic factors and relative survival. Ann Oncol\n19: 276–28317962200\n4. Ernst MF, van de Poll-Franse LV, Roukema JA, Coebergh JW, van Gestel CM, Vreugdenhil G, Louwman MJ, Voogd AC (2007) Trends in the prognosis of patients with primary metastatic breast cancer diagnosed between 1975 and 2002. Breast\n16: 344–35117303426\n5. Grunfeld E, Noorani H, McGahan L, Paszat L, Coyle D, van Walraven C, Joyce J, Sawka C (2002) Surveillance mammography after treatment of primary breast cancer: a systematic review. Breast\n11: 228–23514965672\n6. Khatcheressian JL, Wolff AC, Smith TJ, Grunfeld E, Muss HB, Vogel VG, Halberg F, Somerfield MR, Davidson NE (2006) American Society of Clinical Oncology 2006 update of the breast cancer follow-up and management guidelines in the adjuvant setting. J Clin Oncol\n24: 5091–509717033037\n7. Kurtz JM, Amalric R, Brandone H, Ayme Y, Spitalier JM (1988) Contralateral breast cancer and other second malignancies in patients treated by breast-conserving therapy with radiation. Int J Radiat Oncol Biol Phys\n15: 277–2843136104\n8. Lash TL, Fox MP, Buist DS, Wei F, Field TS, Frost FJ, Geiger AM, Quinn VP, Yood MU, Silliman RA (2007) Mammography surveillance and mortality in older breast cancer survivors. J Clin Oncol\n25: 3001–300617548838\n9. Montgomery DA, Krupa K, Jack WJ, Kerr GR, Kunkler IH, Thomas J, Dixon JM (2007) Changing pattern of the detection of locoregional relapse in breast cancer: the Edinburgh experience. Br J Cancer\n96: 1802–180717533401\n10. National Institute for Clinical Excellence (2002) Guidance on cancer services: Improving Outcomes in Breast Cancer -Manual update. www.nice.org.uk (accessed 22nd November 2007)\n11. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin\n55: 74–10815761078\n12. Rack B, Janni W, Gerber B, Strobl B, Schindlbeck C, Klanner E, Rammel G, Sommer H, Dimpfl T, Friese K (2003) Patients with recurrent breast cancer: does the primary axillary lymph node status predict more aggressive tumor progression? Breast Cancer Res Treat\n82: 83–9214692652\n13. Schootman M, Fuortes L, Aft R (2006) Prognosis of metachronous contralateral breast cancer according to stage at diagnosis: the importance of early detection. Breast Cancer Res Treat\n99: 91–9516555124\n14. Smith TJ, Davidson NE, Schapira DV, Grunfeld E, Muss HB, Vogel III VG, Somerfield MR (1999) American Society of Clinical Oncology 1998 update of recommended breast cancer surveillance guidelines. J Clin Oncol\n17: 1080–108210071303\n15. Tabar L, Fagerberg G, Duffy SW, Day NE, Gad A, Grontoft O (1992) Update of the Swedish two-county program of mammographic screening for breast cancer. Radiol Clin N Amer\n30(1): 187–2101732926\n16. The Association of Breast Surgery @ BASO, Royal College of Surgeons of England (2005) Guidelines for the management of symptomatic breast disease. Eur J Surg Oncol\n31(Suppl 1): 1–21\n17. van der Sangen MJ, van de Poll-Franse LV, Roumen RM, Rutten HJ, Coebergh JW, Vreugdenhil G, Voogd AC (2006) The prognosis of patients with local recurrence more than five years after breast conservation therapy for invasive breast carcinoma. Eur J Surg Oncol\n32: 34–3816305821"
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+{
+    "id": "PMC2527820",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527820\nAUTHORS: B M Beckermann, G Kallifatidis, A Groth, D Frommhold, A Apel, J Mattern, A V Salnikov, G Moldenhauer, W Wagner, A Diehlmann, R Saffrich, M Schubert, A D Ho, N Giese, M W Büchler, H Friess, P Büchler, I Herr\n\nABSTRACT:\nLittle is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31+ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.\n\nBODY:\nThe plastic adherent cells from the bone marrow (BM) referred to as mesenchymal stem cells (MSC) are capable of self-renewing and have the potential to differentiate into mesenchymal and non-mesenchymal tissues (Prockop, 1997). Mesenchymal stem cells contribute to tissue regeneration by differentiation into bone, cartilage, muscle, ligament, tendon, adipose tissue, and stroma (Pittenger et al, 1999). The ability of MSC to migrate to the areas of injury and to tumours has encouraged investigation of MSC as therapeutic tools. For example, systemically administered MSCs have been shown to improve recovery in animal models of stroke and myocardial infarction (Mahmood et al, 2003; Fukuda and Fujita, 2005). Mesenchymal stem cells have also been used for targeted delivery of therapeutic gene products to the tumour microenvironment in animal models (Studeny et al, 2002, 2004; Nakamizo et al, 2005). This shared tropism of MSC for sites of injured tissue and for tumours is believed to result from similarities in the inflammatory milieu produced by healing wounds and tumours, evoking the notion that ‘tumours are wounds that never heal’ (Dvorak, 1986). Furthermore, a role of MSC in neoangiogenesis is discussed, as the administration of MSC stimulated revascularisation of ischaemic tissues (Chen et al, 2003; Nagaya et al, 2004). A correlation between angiogenesis in melanoma and MSC has recently been demonstrated (Sun et al, 2005), and MSC have been found to transmigrate over the endothelial barrier (Schmidt et al, 2006b). In all cases, MSCs had to cover a distance to reach the target area. Therefore, the common hypothesis is that MSC possess a migratory activity. The most prominent chemotactic factors identified thus far for MSC include SDF-1, basic fibroblast growth factor and vascular endothelial growth factor (VEGF) (Schmidt et al, 2006a). The concept of chemokines as possible chemoattractants for MSC may be of importance for pancreatic cancer, as this tumour entity contains poorly vascularised regions characterised by severe hypoxia resulting in the expression of growth factors (Bos et al, 2005; Patiar and Harris, 2006).It has been suggested that the MSC-mediated effects can be attributed, at least in part, to the biologically active factors secreted by MSC itself at their target sites. Even more, a direct contribution of MSC to the blood vessel formation is suggested, as differentiation of MSC into endothelial cells has been demonstrated (Oswald et al, 2004; Silva et al, 2005; Song et al, 2007), although this concept remains controversial. Here, we test the hypothesis that hypoxia-induced growth factor expression in pancreatic cancer promotes tumour angiogenesis by mediating MSC recruitment.We assessed this hypothesis by in vitro studies and in an orthotopic mouse model of pancreatic carcinoma. We demonstrate the migration of MSC towards growing normal and tumour cells, as well as to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and vascular epidermal growth factor (VEGF). Inhibitors of PDGFR (Glivec), EGFR (Erbitux) and blocking antibody to VEGF (Avastin) interfered with MSC migration demonstrating the specific growth factor-mediated effect. Within a few hours, MSC migrated into pancreatic tumour cell spheroids as measured by time-lapse microscopy. Mesenchymal stem cells themselves secreted VEGF, and the transfer of supernatant from cultured MSC induced sprouting of endothelial cells. Differentiation of MSC to endothelial cells was observed in only few cells in vitro but not in vivo. However, MSC inoculated in nude mice xenografted with orthotopic pancreatic tumours were found to home into the tumours and to incorporate in tumour blood vessels. Our conclusion is that MSC contribute to the tumour blood vessel formation by homing to fast growing tumours and the incorporation into blood vessels as atypical VEGF-secreting endothelial cells.Materials and methodsIsolation and culture of human BM-derived MSCMesenchymal stem cells were isolated from human BM of healthy donors, selected by plastic adherence, and were cultivated as described in our recent publication (Kallifatidis et al, 2008).Primary and established human cell linesEstablished human standard cancer cell lines of pancreas (Capan-1, MIA-Pa-Ca2, Colo-357, and BxPC-3), immortalised human kidney cells (HEK 293T), and human primary fibroblasts from the skin (kind gift from J Knebel and P Angel) were cultured in DMEM supplemented with 10% FCS. Human umbilical vein endothelial cells (HUVEC; PromoCell, Heidelberg, Germany) were cultured in endothelial cell growth medium (ECGM; PromoCell).Migration of MSC to chemoattractants measured by transwell chamber migration assayA ChemoTx® System (Neuro Probe Inc., Gaithersburg, MD, USA) with 96 wells or Transwell® permeable supports (Corning Incorporated, Life Sciences, Acton, MA, USA) and a polycarbonate membrane pore size of 12 μm was used. The bottom chamber contained PDGF (R&D Systems, Wiesbaden-Nordenstadt, Germany), EGF (R&D Systems), or VEGF (BioSource, Nivelles, Belgium) in medium with 2% FCS or cell culture supernatant of tumour cells grown in medium supplemented with 2% FCS for 2 days. Growth factors were used with or without specific inhibitors: Avastin for the inhibition of VEGF (25 μg ml−1; Roche, Welwyn Garden City, Hertfordshire, UK), Glivec for the inhibition of PDGF receptors (3 μM; Novartis, Horsham, West Sussex, UK), and Erbitux for the inhibition of EGF receptors (3 μM; Merck, Darmstadt, Germany). The migration of MSC was analysed as described in our recent publication (Kallifatidis et al, 2008).Generation of tumour cell spheroidsSpheroids were generated as described (Korff et al, 2004). Confluent monolayers of MIA-PaCa-2 cells, primary fibroblasts, and HUVEC were trypsinised. A total of 500 MIA-PaCa-2 cells, 250 fibroblasts, and 250 HUVECs per spheroid were mixed in corresponding culture medium containing 0.25% (w/v) methylcellulose (Sigma, Steinheim, Germany) and seeded in nonadherent round bottom 96-well plates (Greiner, Frickenhausen, Germany). Under these conditions, all suspended cells contribute to the formation of a single spheroid per well of defined size and cell number. Spheroids were cultured for at least 24 h and used for the corresponding assay.Migration of MSC to tumour cell spheroidsMesenchymal stem cells were labelled with CellTracker Red according to the instructions of the manufacturer (Molecular Probes, Eugene, OA, USA). A total of 1 × 104 MSCs (in a volume of 225 μl) were seeded in one edge of a fibronectin-coated well of a 24-well plate by canting the plate in an angle of 30°. After 24 h, MSC culture medium was removed, and cells were covered with 225 μl of methylcellulose/collagen solution (40% methylcellulose stock solution, 10% FCS, and 50% collagen solution prepared from rat tail). Correspondingly, spheroids were labelled with Cell Tracker Green (Molecular Probes), and 48 spheroids were seeded opposite to the MSC in the same well. Invasion of MSC in spheroids was documented by time-lapse microscopy and a camera with a red filter and green filter. The camera was focused to the green fluorescent spheroids and cells were observed over 12 h on an Olympus IX70 microscope equipped with an incubation housing around the microscope. Time-lapse imaging series were acquired using the software analySiS from Soft Imaging System with a ColorView-12 digital colour camera. Images were made every 5 min, for each position one with bright field and one with fluorescence illumination in red and/or green with a motorised Ludl X-Y microscope stage.In vitro angiogenesis assaySpheroids containing 750–1000 HUVECs were generated overnight, after which they were embedded in collagen gel as described previously (Korff et al, 2001). Supernantant of MSC or recombinant VEGF (BioSource) were transferred to the spheroids. After 24 h, in vitro angiogenesis was digitally quantified by measuring the length of the sprouts that had grown out of each spheroid (at × 10 magnification) using the digital imaging software cellB 2.3 (Olympus, Hamburg, Germany) analysing at least eight spheroids per experimental group and experiment.Detection of VEGF and α-smooth muscle actin in MSC by RT-PCRMesenchymal stem cells were seeded in a six-well plate at a concentration of 1 × 105 cells per well and incubated overnight under normoxic conditions (37°C, 5% CO2) followed by 16 h incubation in a hypoxia chamber (N2: 89.25%, CO2: 10%, and O2: 0.75%). Cells were scraped in 300 μl per well prewarmed Magna Pure LC Lysis buffer (mRNA isolation kit I for cells; Roche Applied Science, RAS, Mannheim, Germany) supplemented with 0.01 g ml−1 DTT from the tissue culture plates and frozen at −80°C. mRNA/cDNA preparation and real-time quantitative PCR were performed with equipment and reagents from Roche as described previously (Erkan et al, 2007). In brief, mRNA was extracted by automated isolation, and cDNA was prepared using the First Strand cDNA synthesis kit for RT-PCR (ABgene Advanced Biotechnologie, Epsom, UK). QRT-PCR was performed using LightCycler™ primer sets obtained from Search-LC (Heidelberg, Germany). Results are expressed as the number of VEGF or α-SMA transcripts per 10 000 CPB transcripts (cyclophilin B, a housekeeping gene).Lentiviral transduction of MSCVectors used in our study are the self-inactivating (SIN) vectors of the second generation, which loose the activity of the promoter located in the 5′-LTR upon replication and integration into the genome of the host cells. The construction of the pLL3.7puroeGFP vector plasmid, VEGF siRNA, and lentiviral transduction of MSC is described in our recent manuscript (Kallifatidis et al, 2008). In detail, the VEGF siRNA sequence was designed against the human VEGF mRNA (accession no. AF022375), the target sequence starting at position 465 after the start codon. Depicted is the resulting VEGF siRNA sequence with polyA tail, siRNA stem, loop and siRNA stem: 5′-tcgagaaaaaagatccgcagacgtgtaaatgtctcttgaacatttacacgtctgcggatca-3′. The construct was verified by sequencing.Detection of VEGF in cell culture supernatant of MSCA volume of 5 × 104/ml lentiviral transduced or non-transduced MSC were seeded in 12-well plates. Hypoxia was induced as described above, and 16 h later, cells were quickly removed from hypoxic conditions by putting them on ice. The VEGF content was estimated in supernatant and cell lysates following the instructions of the Quantikine® ELISA for human VEGF (R&D Systems, Wiesbaden-Nordenstadt, Germany). Cells treated with 25 μg ml−1 Avastin added at the time of hypoxia induction served as negative control. Vascular endothelial growth factor expression was evaluated in an ELISA plate reader at 450 nm with a correction at 570 nm. Results were normalised to picogram VEGF per hour treatment per 104 cells.In vitro differentiation of MSC in endothelial cellsMesenchymal stem cells (1 × 104/cm2) were seeded in a six-well plate, and for differentiation, 50 ng ml−1 VEGF (Biosource, Nivelles, Belgium) was added to standard culture medium or to ECGM used for HUVEC culture. Differentiation to endothelial cells was analysed by using the Chemicon (Temecula, CA, USA) blood vessel staining kit following supplier's instructions. Shortly, the cells were incubated with rabbit anti-vWF polyclonal antibody (1 : 200, Chemicon) or mouse anti-CD31 monoclonal antibody (1 : 200, Chemicon) and detected with biotinylated goat anti-rabbit or goat anti-mouse antibody and Streptavidin-HRP (Chemicon). DAB/haematoxylin staining was performed by a standard protocol. Cells were analysed with a Leica DMRB microscope (Leica Microsystems GmbH, Wetzlar, Germany) with Kappa CF20/4 DX Camera (Kappa Opto-Electronics GmbH, Gleichen, Germany).Detection of microvessel density in xenograftsTo examine the effects of MSC injection on the microvessel density in xenografts, aceton-fixed frozen sections (5 μm) were stained with rat anti-mouse CD31 mAb (PharMingen, San Diego, CA, USA) as described previously (30). Random areas of tumours were then examined under higher magnification ( × 250) and CD31+ structures were counted. Any distinct area of positive staining for CD31 was counted as a single vessel. Results were expressed as the mean number of vessels±s.e. per mm2. A total of eight high-power fields was examined and counted from four tumours of each of the treatment groups.Orthotopic pancreatic cancer xenograft model in athymic nude miceNMRI (nu/nu) male mice (6-to-10-weeks old) were used for subcutaneous and orthotopic tumour implantations of the human pancreatic cancer cell line MIA-PaCa-2 as described previously (Buchler et al, 2007). The experimental protocol was approved by the Chancellor's Animal Research Committee of the University of Heidelberg (Heidelberg, Germany) in accordance with National Guidelines for Animal Care and the Use of Laboratory Animals.Detection of GFP-MSC in blood vessels of pancreatic cancer xenograftsCryosections were fixed in 4% PFA for 15 min and permeabilised in 0.2% Triton X-100 for 15 min. The sections were incubated with mouse anti-eGFP antibody 1 : 200 (JL8, BD Clontech, Heidelberg, Germany) in PBS/5% goat serum and detected with goat anti-mouse FITC-conjugated antibody (Invitrogen, Karlsruhe, Germany) diluted 1 : 200 in PBS/5% goat serum. For secondary staining, sections were incubated with rabbit anti-vWF antibody 1 : 200 (Chemicon) in PBS/5% goat serum and detected with biotinylated goat anti-rabbit Ab 1 : 200 (KPL, Gaithersburg, MD, USA) and Texas Red Avidin 1 : 200 (Vector Laboratories, Peterborough, UK).Intravital microscopy of GFP-MSC recruitment in miceAnaesthesia, general preparation, surgical preparation of the cremaster muscle and xenograft, intravital microscopy, and data analysis were performed as previously described (Sperandio et al, 2001, 2006). Briefly, after anaesthesia, the trachea was intubated, and the left carotid artery was cannulated with PE 10 tubing (ID: 0.28 mm, OD: 0.61 mm; Becton Dickinson) for the administration of MSC and anaesthetics throughout the intravital microscopic experiment. For prevention of ischaemic events, the number of injected cells was restricted to 1 × 105 MSC/0.2 ml normal saline per application up to a total number of 4 × 105 MSC during 1 h. Intravital microscopy was conducted on an upright microscope (Leitz, Wetzlar, Germany) with a saline immersion objective (SW 40/0.75 numerical aperture) and epifluorescence illumination (60/s; Strobex 236, Chadwick Helmuth, Mountain View, CA, USA; and filter block Zeiss 9). After recording, the resulting video was digitalised to MPEG format using Thyphoon DVD maker and TVR software (ANUBIS Electronic Ltd., Kowloon, Hong Kong).Statistical analysisData are presented as the mean±s.e. For in vitro experiments, Student's t-test was used to evaluate the differences between groups. For in vivo experiments, Mann–Whitney U-test was used to evaluate the differences between groups. In both calculations, P<0.05 was considered statistically significant.ResultsIsolation and expansion of human MSCBone marrow aspirates were obtained from normal human donors, isolated, and expanded according to our recent publication (Kallifatidis et al, 2008). Cells had a typical spindle shape, consistent with the morphology reported by others (Pittenger and Marshak, 2001). Although MSC do not have a specific antigen profile, we verified for each culture that isolated cells were negative for typical haematopoietic antigens CD45, CD34, and CD38 and were positive for CD44 and CD105 (data not shown). The doubling time of our cultures varied between 30 and 40 h, and cells could be expanded to 8–12 passages before reaching senescence. Thus, based on available criteria, the cells used in our experiments had the properties of human MSC as described.Specific migration of MSC to PDGF, EGF, and VEGFFactors released by cancer cells may be potential mediators of MSC migration. To test this hypothesis, we performed in vitro migration assays using Transwell plates to evaluate the tropism of human MSC for cancer cells. We first investigated if human established pancreatic cancer cell lines were capable of stimulating the migration of MSC. Normal cells, such as T293, primary fibroblasts, and endothelial cells, were also investigated. Mesenchymal stem cells were placed in the upper wells, and conditioned medium from cells grown in medium with 2% FCS was placed in the lower wells. Cell-free medium with 20 or 2% FCS was used as positive and negative controls, respectively. A semiporous membrane (12 μm pores) separated the wells. Migration was quantified by directly visualising and counting migrated cells under the microscope after cell staining. Exposure to cell-free medium with 20% FCS and to all cell-conditioned media resulted in significant migration of MSC, when compared with cell-free medium with 2% FCS (Figure 1A). The observed differences in migration were not due to the increase in MSC proliferation because the total number of MSC (migrating plus nonmigrating) was the same for each condition. As 20% FCS and conditioned medium from growing cells stimulated significant MSC migration, it is plausible that growth factors may be chemoattractants for MSC. Therefore, to analyse the role of growth factors in MSC migration, PDGF, EGF, or VEGF was added to the lower wells. Maximal MSC migration occurred with exposure to PDGF. Intermediate levels of migration were observed after exposure to EGF followed by VEGF, which had a significant effect compared with control medium with 2% FCS (Figure 1B). To document that the increase in migration of MSC was specifically due to the presence of growth factors, inhibitors of PDGFR (Glivec), EGFR (Erbitux), and VEGF (Avastin) were added together with growth factors. These blocking substances prevented the activity of the respective growth factors (Figure 1C), suggesting specific migration of MSC to growth factors. As migration of MSC towards VEGF is the link to tumour angiogenesis, we examined VEGF expression by pancreatic cancer cells. Because VEGF is under the control of the transcription factor HIF-1α, which is induced by tumour hypoxia, we performed western blot experiments using BxPc-3 cells grown under normoxic and hypoxic conditions. Strong induction of HIF-1α was observed as early as 2 h after hypoxia, which lasted for 16 h and dropped down to basal levels after 24 h (Figure 1D). In parallel, BxPc-3 cells secreted VEGF into the supernatant, which could be completely blocked by adding Avastin to the cell culture medium as measured by an ELISA assay. Thus, it appears that enhanced levels of VEGF and other growth factors secreted by pancreatic cancer cells under hypoxic conditions lead to the migration of MSC.MSC are attracted by reconstructs of tumour blood vesselsTo examine, whether MSC may be attracted to tumour blood vessel reconstructs, we created tumour cell spheroids. These consisted of MIA-PaCa-2 pancreatic cancer cells, primary fibroblasts, and HUVECs. Pancreatic cancer cells and fibroblasts are known to overexpress many growth factors (Korc, 2007), including VEGF (Sipos et al, 2002). This, in turn, may lead to the paracrine stimulation of PDGF expression in HUVECs as recently described (Reinmuth et al, 2007). Therefore, tumour cell spheroids may be strong chemoattractants for MSC. To prove this hypothesis, green fluorescent spheroids and red fluorescent MSC were seeded opposite in wells of a 24-well plate and separated by a methylcellulose/collagen solution (Figure 2A). The invasion of MSC into spheroids was documented by time-lapse microscopy over 12 h focused on the green fluorescent spheroids. Within 1 h, red MSC came into the focus, indicating that MSC migrate to the spheroids (Figure 2B). Migration was completed within 5 h. These results suggest the migration of MSC to pancreatic tumour spheroids.MSC induce sprouting but do not differentiate into endothelial cells in vitroTo investigate the effects of paracrine factors secreted by MSC on angiogenesis, we measured VEGF expression in MSC cultured under normoxic and hypoxic conditions. The secretion of VEGF in cell culture supernatant was analysed by the ELISA assay and the expression of RNA by RT-PCR. By this way, we found that MSC express a basal level of VEGF protein and RNA, which could be enhanced by hypoxia (Figure 3A and B). Total blocking of basal and induced VEGF expression by Avastin served as control. To see whether the MSC-secreted VEGF is able to contribute to angiogenesis, we added MSC supernatant to HUVEC. This resulted in strong induction of sprouting in similar intensity as observed with the addition of recombinant VEGF alone (Figure 3C). Owing to this obvious angiogenic potential of MSC, we examined the differentiation of MSC into endothelial cells by culturing the MSC for 1, 2, or 3 weeks in endothelial cell culture medium containing VEGF. However, even after prolonged time, we could detect the expression of typical endothelial cell markers, such as CD31 or von Willebrand factor only in very few (about 0.01%) MSC (Figure 3D). In contrast, HUVECs, which were used as positive control, strongly expressed these typical markers for endothelial cells. However, 10% of MSC from the same fraction kept their differentiation potential to osteocytes and adipocytes as tested by culture in specific differentiation media as described (data not shown). Therefore, the differentiation of MSC into endothelial cells may be neglected as a main factor responsible for an angiogenic potential of MSC.Migration of MSC in tumour blood vessels and attachment to vessel endotheliumMice with human orthotopic pancreatic cancer xenografts were injected with LV-transduced MSC (4 × 105 in tail vein) conferring the expression of enhanced green fluorescent protein (eGFP). Xenografted mice, which did not receive MSC, served as controls. Three days later, mice were killed followed by resection of xenografts and organs. Isolated cells were examined by flow cytometry for eGFP expression (Figure 4A). The highest eGFP expression was observed in tumour xenografts, but a minor percentage was also detected in normal organs, such as the lung, spleen, and kidney but not in the liver or heart. Weak green fluorescence was observed in tumours of mice that did not receive MSC, suggesting autofluorescence of the mouse tissue. This result indicates a tumour-specific migration of MSC and demonstrates that few MSC reside in other organs according to a natural function of MSC in tissue remodelling (Jiang et al, 2002). To see whether MSC influence tumour angiogenesis, we injected MSC with lentiviral control vector or with lentiviral siRNA into mice with orthotopic MIA-PaCa-2 xenografts. Three days later, cryosections of xenografts were examined for the expression of CD31 to detect blood vessels. To quantify the density of blood vessels, we counted CD31+ structures per high-power field ( × 250) (Figure 4B and C). By this way, we found that MSC with empty vector lead to a doubling of blood vessels in contrast to mice that received no MSC or MSC with siRNA-inhibited VEGF expression. As siVEGF lead to a strong inhibition of VEGF protein secretion in transduced MSC (Figure 4D), the production of VEGF by MSC may be a major reason for the angiogenic capacity of MSC.Intravital microscopy of MSC recruitment in miceTo further elucidate the process of MSC homing in vivo, we monitored microcirculation in cremaster muscle and in orthotopic Mia-PaCa-2 xenograft tumours using fluorescence intravital microscopy immediately after intraarterial injection of MSC expressing eGFP. Within 5–10 s after the injection of MSC, the green fluorescent cells appeared in the inflamed cremasteric microcirculation, first freely flowing in small arteries and arterioles, and later in the capillary network where some of them got already stuck. As soon as 1 min after MSC administration, eGFP+ cells were detectable in the cremasteric venular tree (Figure 5A, Supplementary Figure S1). We observed firm arrest of individual MSC in cremasteric venules and veins (vessel diameter 120 μm, centerline blood flow velocity of 2100 μm s−1 with a resulting wall shear rate of 700 s−1). Although technically more challenging, we were able to observe MSC homing in the xenograft tumours similar to MSC recruitment in cremaster muscle. After arterial circulation, some MSC adhered in tumour capillaries and later also in tumour venules and veins (vessel diameter 45 μm, centerline blood flow velocity of 800 μm/s with a resulting wall shear rate of 700 s−1) (Figure 5B, Supplementary Figure S2). A few of the observed MSC detached 30 s after initial arrest. The attachment of MSC to vascular endothelium was confirmed by immunofluorescence staining of an eGFP-expressing MSC within vWF-positive endothelial cells within a tumour vessel of a pancreatic cancer xenograft (Figure 5C).DiscussionCellular therapy with human MSC has great potential for use in regenerative medicine and is currently in clinical development. Mesenchymal stem cells are being investigated in the treatment of bone and cartilage defects, and injured myocardium after acute infarction (Giordano et al, 2007). The ability of MSC to differentiate into several lineages of connective tissue is well documented (Prockop, 1997). Recent evidence has suggested that MSC may also differentiate into endothelial and vascular smooth muscle cells (Oswald et al, 2004). In this report, we set out to clarify molecular mechanisms of MSC migration towards tumour blood vessels and the cellular fate of implanted MSC in participating in vasculogenesis in vivo. Our study shows the migration of MSC towards growth factors secreted by growing tumour and normal cells, with PDGF as most potent chemoattractant, followed by EGF and VEGF, in line with recent findings (Nakamizo et al, 2005; Schmidt et al, 2006a). Moreover, we found the secretion of VEGF by MSC itself under basal conditions and the enhancement of VEGF mRNA and protein secretion into the supernatant by hypoxia. As expected, the transfer of supernatant from MSC to HUVEC increased sprouting, supporting the angiogenic potential of MSC. Therefore, VEGF production by MSC may be a crucial factor responsible for an angiogenic potential of MSC. Another reason for contribution of MSC to angiogenesis may be the differentiation of MSC in endothelial cells as recently suggested (Oswald et al, 2004; Silva et al, 2005; Song et al, 2007). As these data are unclear, we tried to differentiate MSC into endothelial cells by culturing them in endothelial cell culture medium with VEGF. However, we found the differentiation of only few MSC below 0.01% into cells expressing vWF, a typical marker of vascular endothelial cells. The majority of MSC remained undifferentiated under these conditions, although the differentiation potential into adipocytes and osteocytes was intact in the same fractions. The reason for this low differentiation capacity into endothelial cells may be of experimental nature due to suboptimal growth conditions for MSC in endothelial cell culture medium. After 3 weeks in this medium, the MSC resembled rather senescent cells than endothelial cells. However, the differentiation capacity of MSC into endothelial cells is underlined by other reports. In 1999, Barry et al (1999) identified the endothelial marker endoglin (CD105) in BM-derived MSC grown on an endothelial cell-specific membrane. Although undifferentiated BM-derived MSC did not express vWF (Hu et al, 2003), expression of this and some molecules specific to endothelial cells was found after endothelial differentiation on the cell surface of MSC (Oswald et al, 2004; Sun et al, 2005). But blood vessels are not only composed of endothelial cells. Endothelial cells constitute the inner lining of the vessel wall, and pericytes and vascular smooth muscle cells (summarised as mural cells) envelop the surface of the vascular tube in mature, stable vessels. During the early stage of angiogenesis, neovascular sprouts are composed primarily of endothelial cells and are subsequently stabilised by recruiting mural cells (Jain, 2003). Human MSC implanted in immunodeficient mice recruit into blood vessels as shown by our immunohistochemistry and microcirculation studies. However, we did not detect in vivo differentiation of MSC into endothelial cells and the function of MSC in endothelium is unclear so far. In related models, it has been described that co-implantation of HUVEC with a murine mesenchymal precursor cell line (10T1/2) that differentiates into mural cells is necessary for the formation of long-lasting blood vessels (Koike et al, 2004). In consequence, MSC may be a source for mural cells or be involved in recruitment of them. A recent publication points to this direction, as co-implantation of HUVEC and human MSC in immunodeficient mice was able to form human mature blood vessels, which are stable and functional for more than 3 months (Sanz et al, 2008). Another recent study suggested MSC as perivascular cell precursors and MSC seem to contribute to a functional vasculature by differentiation into pericytes (Au et al, 2008). In vivo, we detected homing of MSC to tumour blood vessels of an orthotopic pancreatic carcinoma xenograft in immunodeficient mice. After injection of MSC in tail vein, we found enhanced vessel density by control MSC but not by MSC expressing siRNA directed towards VEGF. Therefore, our data suggest that VEGF production by MSC is the critical factor mediating the angiogenic potential of MSC. Tumour hypoxia, a feature typical for pancreatic cancer may further increase the angiogenic effect of MSC, as our in vitro data show the expression of HIF-1α by pancreatic cancer cells under hypoxic conditions, which, in turn, leads to expression and secretion of VEGF by tumour cells. Enhanced VEGF levels may increase migration of MSC, which, after recruitment to pancreatic tumours, itself produce VEGF and contribute by this way to the hypoxia-initiated angiogenic effect.For in vivo detection of MSC recruitment in trauma-induced inflammation of cremaster muscle and orthotopic MIA-PaCa-2 xenograft tumours, we used intravital microscopy. By this way, we found a very early arterial circulation of injected MSC followed by a final arrest of some MSC in the capillary network, which is likely to be due to obstruction. Other MSC were detected in the venular tree as early as 1 min after MSC administration where some of them adhered firmly to the endothelium. Only very few transiently attached MSC detached again from the vessel wall. Although there are some rolling MSC detectable, the rather sudden arrest of MSC seems to be similar to the VCAM-1-dependent fashion of leukocyte adhesion triggered by VLA-4 (Ley et al, 2007). As VLA-4 is known to be expressed on MSC and VCAM-1 on inflamed vascular endothelium (Ley et al, 2007), one may hypothesise an in vivo relevance of the interaction of these two molecules during MSC recruitment as supported by recent studies (Ruster et al, 2006; Segers et al, 2006). Furthermore, similar to leukocytes, MSC seem to be recruited preferentially in low flow (capillaries) or medium flow (venules) vessels providing optimal haemodynamic conditions, that is, wall shear rate (Long et al, 2004). With these respects to leukocyte recruitment, further elucidation of exact mechanisms of the early steps of MSC recruitment into both peripheral tissue and neoplasic tumours is required.In conclusion, we demonstrate that human MSC from BM specifically migrate to tumour blood vessels of pancreatic carcinoma in vitro and in vivo, which can be attributed to tumour hypoxia-induced secretion of VEGF and other growth factors. We observed long-term (3 days) interaction of MSC with the endothelium of tumour blood vessels. Very few of MSC differentiated into endothelial cells in vitro, whereas no differentiation in vivo could be observed. We found high VEGF production by MSC, which was further enhanced by hypoxia. 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Schmidt A, Ladage D, Steingen C, Brixius K, Schinkothe T, Klinz FJ, Schwinger RH, Mehlhorn U, Bloch W (2006b) Mesenchymal stem cells transmigrate over the endothelial barrier. Eur J Cell Biol\n85: 1179–118816824647\n33. Segers VF, Van Riet I, Andries LJ, Lemmens K, Demolder MJ, De Becker AJ, Kockx MM, De Keulenaer GW (2006) Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms. Am J Physiol Heart Circ Physiol\n290: H1370–H137716243916\n34. Silva GV, Litovsky S, Assad JA, Sousa AL, Martin BJ, Vela D, Coulter SC, Lin J, Ober J, Vaughn WK, Branco RV, Oliveira EM, He R, Geng YJ, Willerson JT, Perin EC (2005) Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation\n111: 150–15615642764\n35. 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Blood\n97: 3812–381911389021\n39. Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M (2002) Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res\n62: 3603–360812097260\n40. Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN, Champlin RE, Andreeff M (2004) Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst\n96: 1593–160315523088\n41. Sun B, Zhang S, Ni C, Zhang D, Liu Y, Zhang W, Zhao X, Zhao C, Shi M (2005) Correlation between melanoma angiogenesis and the mesenchymal stem cells and endothelial progenitor cells derived from bone marrow. Stem Cells Dev\n14: 292–29815969624"
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+    "id": "PMC2527825",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527825\nAUTHORS: O J Vilholm, S Cold, L Rasmussen, S H Sindrup\n\nABSTRACT:\nThe prevalence of the postmastectomy pain syndrome (PMPS) and its clinical characteristics was assessed in a group of patients who had undergone surgery for breast cancer at the Department of Surgery, Odense University Hospital, within the period of 1 May 2003 to 30 April 2004. The study included 258 patients and a reference group of 774 women. A questionnaire was mailed to the patients 1½ year after surgery and to the women in the reference group. The PMPS was defined as pain located in the area of the surgery or ipsilateral arm, present at least 4 days per week and with an average intensity of at least 3 on a numeric rating scale from 0 to 10. The prevalence of PMPS was found to be 23.9%. The odds ratio of developing PMPS was 2.88 (95% confidence interval 1.84–4.51). Significant risk factors were as follows: having undergone breast surgery earlier (OR 8.12), tumour located in the upper lateral quarter (OR 6.48) and young age (OR 1.04). This study shows that, although recent advances in the diagnostic and surgical procedures have reduced the frequency of the more invasive surgical procedures, there still is a considerable risk of developing PMPS after treatment of breast cancer.\n\nBODY:\nWithin the last decade, it has been increasingly accepted that chronic pain can be a complication to surgery (Perkins and Kehlet, 2000; Macrae, 2001). Chronic pain has been reported after a range of different types of surgery, such as thoracotomy, inguinal hernia repair and hysterectomy.Similarly, the prevalence of chronic pain after surgery for breast cancer, the postmastectomy pain syndrome (PMPS), has been known to develop in 20–68% of patients (Ivens et al, 1992; Maunsell et al, 1993; Stevens et al, 1995; Tasmuth et al, 1995; Wallace et al, 1996; Carpenter et al, 1998; Smith et al, 1999; Kuehn et al, 2000; Peintinger et al, 2003).The description, the postmastectomy pain syndrome, is somewhat misleading, as the syndrome also includes chronic pain after breast conserving surgery. The exact mechanism of development is not known, but it is probably a neuropathic pain condition, which is because of damage to the nerves in the axilla and/or the chest wall during surgery. On this basis, an alternative term, intercostobrachial neuralgia, has been proposed (Jung et al, 2003). PMPS can develop shortly after surgery or up to several months after surgery and can persist for years. Other causes for chronic pain after treatment for breast cancer include intercostal neuromas (Rosso et al, 2000; Wong, 2001; Kretschmer et al, 2002).The prevalence of PMPS has been shown to be higher after lumpectomy than after mastectomy (Tasmuth et al, 1996, 1997). The pain is often located in the axilla, the shoulder, the arm or the chest wall. PMPS is often described as a typical neuropathic pain consisting of burning pain, shooting pain, pain evoked by pressure and deep blunt pain. Like other neuropathic pain conditions, the treatment is often difficult. A study from 1994 and a recent study suggest that the prognosis of PMPS is better than expected, with a decline in prevalence over years (de Vries et al, 1994; Macdonald et al, 2005).Previous studies have identified following risk factors: young age (Tasmuth et al, 1995; Smith et al, 1999; Poleshuck et al, 2006), sectioning of the intercostobrachial nerve (Abdullah et al, 1998; Torresan et al, 2003) and axillary dissection (Vecht, 1990; Maunsell et al, 1993; Hack et al, 1999; Kakuda et al, 1999; Johansen et al, 2000). Dissection of axillary lymph nodes has been shown to be a critical component in the aetiology of chronic pain after surgery for breast cancer. The frequency of this procedure has been reduced over the past few years, due to the introduction of the sentinel node biopsy (Shons and Cox, 2001). The morbidity of this procedure has been shown to be less than standard axillary treatment (Kakuda et al, 1999; Schijven et al, 2003; Mansel et al, 2006). Thus, the prevalence of PMPS may have decreased (Miguel et al, 2001). This study estimates the current prevalence of PMPS and identifies risk factors.Materials and methodsA postal survey concerning pain after surgery for breast cancer was undertaken in the County of Funen, Denmark. Invitations to participate, along with questionnaires, were mailed to a group of breast cancer patients, 1½ year after their surgery and to a group of reference subjects. This time point was chosen to be sure to enrol all chronic pain complaints and to avoid acute and transient postoperative pain complaints. The inclusion criterion was as follows: women who had undergone surgery for breast cancer with either mastectomy or lumpectomy, performed at the Department of Surgery, Odense University Hospital within the period from 1 May 2003 to 30 April 2004. The exclusion criteria were as follows: (a) living outside the County of Funen, (b) having undergone thoracotomy, (c) severe angina pectoris. Women with reoccurrence of breast cancer and women who had undergone breast surgery earlier for cancer or cosmetic reasons were included in the study.The reference group was randomly selected from the same general population as the breast cancer patients. For each breast cancer patient, three reference subjects, who had not undergone surgery for breast cancer, were selected as controls. The reference group was established from the Danish Civil Registry and stratified to the group of breast cancer patients on age and postal code.A questionnaire was developed (available on request from the authors). Questions about pain focused on present pain and pain experienced in the week preceding the assessment. There were also questions about the location, the intensity and the character of the pain as well as the impact of pain on daily living, the consumption of analgesics and demographic questions. The questionnaires were tested for comments on colleagues and controls. Non-responders received a reminder 1 month after the first mailing. If needed, participants were contacted by telephone for clarifying responses. The Ethical Committee of Vejle and Funen County (VF 20040191) and the Danish Data Protection Agency (J.nr. 2004-41-4514) approved the study, which was performed according to the Declaration of Helsinki.Information about the surgery and the postoperative treatment for the group of breast cancer patients was obtained from the DBCG registry (Danish Breast Cancer Cooperative Group), the Department of Surgery and the Department of Oncology, Odense University Hospital. Comparisons regarding demographic characteristics were conducted using χ2 or Fisher's exact test for categorical data, and non-parametric Mann–Whitney U-test was used for non-categorical data. Univariate logistic regression analysis was applied for testing the possible risk factors. Afterwards, multiple regression analysis was applied for the analysis of possible risk factors, and a priori the following variables were included: age, chemotherapy, radiation therapy, mastectomy, tumour located in the upper lateral quarter. Post hoc, it was decided to add the following variables for the multivariate analysis: having undergone breast surgery earlier, smoking and axillary dissection. These variables were significant or near-significant risk factors in the univariate analysis. Age and tumour size were non-categorical variables. All other variables in the multiple regression analysis were categorical variables. Concerning pain and pain characteristics, odds ratios with 95% confidence interval were calculated. Significance was defined as P<0.05. All analysis was performed with the statistical program STATA version 8. Individuals with missing information from the questionnaire were excluded from the specific analysis.For the group of breast cancer patients, the PMPS was defined as pain located in the area of the surgery or the ispilateral arm, present at least 4 days a week and with an intensity of 3 or more on a numeric rating scale from 0 to 10 (0=no pain and 10=worst possible pain).ResultsQuestionnaires were mailed to 258 breast cancer patients and 774 reference subjects. After one written reminder, 219 (84.9%) of the breast cancer patients and 563 (72.7%) of the reference subjects had returned the questionnaire (Figure 1). The percentages of patients and controls who needed reminders were 16.4 and 15.8%, respectively. The prevalence of PMPS in the group of breast cancer patients was 23.9% and the prevalence of PMPS-like symptoms in the reference group was 10.0%. The odds ratio for developing PMPS after surgery for breast cancer was 2.88 (95% CI 1.84–4.51).Demographic characteristicsThere was no significant difference in age between the responders of the breast cancer patients and the reference subjects (median age being 61.1 (IQR 54.2–68.1) vs 60.3 (IQR 53.7–66.7). In the reference group, the median age was younger for subjects who reported pain similar to PMPS (median age being 55.9 (IQR 49.7–61.8) vs 60.5 (IQR 54.1–66.8) (P=0.003). For other demographic characteristics, there were a higher proportion of subjects from the reference group that had children and twice as many had a family history of chronic pain conditions (Table 1). The characteristics are shown in Table 1. Post hoc analysis showed that the odds ratio for developing PMPS increased to 3.7 (95% CI 1.82–8.30) when adjusting for family history. Positive family history increased the risk of PMPS-like symptoms in both groups. However, as controls were more likely to have a positive family history, this reduces the difference in PMPS-like symptoms between cases and controls.Risk factorsThe results of both univariate and multiple logistic regression analysis are shown in Table 2. When using multiple regression analysis, three significant risk factors were identified: Having undergone breast surgery earlier (OR 8.12), tumour located in the upper lateral quarter (OR 6.48) and young age (OR 1.04). Not-associated risk factors included the following: chemotherapy, axillary dissection, mastectomy, smoking, tumour size and radiation therapy.The location and character of painThe distribution of the location of pain and the character of pain in the group of breast cancer patients with PMPS and in the reference group is shown in Table 1. The majority of the breast cancer patients with PMPS had pain located in the axilla/arm (80.8%) and in the area of the scar (55.8%), and 75.0% of the patients had pain in more than one location. These findings were significantly higher than in the reference group. There were no significant differences with respect to the character of pain.The type of breast surgery, axillary intervention and location of painThe location of pain subdivided according to the type of breast surgery and axillary intervention is shown in Table 3. When applying logistic regression analysis, we found that the odds ratio for developing pain in the mamma was 0.27 (95% CI 0.11–0.72) for lumpectomy, 2.91 (95% CI 0.59–14.4) for sentinel node biopsy and 1.99 (95% CI 0.43–9.20) for axillary dissection. The odds ratio for developing pain in the shoulder, axilla and arm was 0.76 (95% CI 0.36–1.58) for lumpectomy, 0.88 (95% CI 0.25–3.12) for sentinel node biopsy and 1.84 (95% CI 0.58–5.83) for axillary dissection.Taking mastectomy and lumpectomy together, the frequency of pain in general and at any location increased with the extent of axillary intervention (Table 4).Radiation therapy and location of painIn Table 5, the relation between radiation therapy and the location of pain is shown. A higher frequency of pain was observed in the three categories when the radiation therapy included the supraclavicular fields and the axillary region.Systemic therapyNone of the patients received treatment with taxanes or aromatase inhibitors.Pain treatmentThe frequency of consumption of analgesics did not differ significantly between the two groups. Among the breast cancer patients with PMPS, 38.5% reported use of analgesics compared with 52.8% of the reference subjects with pain. The frequency of analgesic consumption was compared between the two groups, the options being ‘Every day’, ‘every other day’, ‘once a week’, ‘every other week’ and ‘once a month’. The distribution of answers in the strata did not differ significantly, P=0.349.The impact of pain on daily lifeMore breast cancer patients than reference subjects reported that pain interfered with daily life ‘really much’ (1.4 vs 0.8%), ‘quite a lot’ (4.6 vs 4.0%), ‘some’ (6.4 vs 1.5%) and ‘a little bit’ (9.2 vs 5.0%). The distribution of answers in the strata differed between the two groups (z=3.52, P=0.0004) (Figure 2).When looking at the patients with pain, who reported pain interfering ‘really much’ or ‘quite a lot’ (n=13), a mean pain score of 7.2 (range 3–10) on a numeric rating scale from 0–10 was found. All 13 patients had pain in the shoulder/axilla/arm and 6 patients had, in addition to this, pain in the mamma. No other common features were found regarding the pain symptoms for this subgroup.DiscussionTo our knowledge, this is the first population-based study of chronic pain after surgery for breast cancer that includes a comparable reference group.The prevalence of PMPS in this study was found to be 24% as compared with PMPS-like symptoms in 10% of the reference group. The odds ratio for developing PMPS after treatment for breast cancer was 2.9. This prevalence for PMPS in women after surgery for breast cancer is similar to results obtained by Smith et al in (1999) (29%) and Carpenter et al in (1998) (27%).In one study, the prevalence has been found to be higher in low volume units than in high volume units (Tasmuth et al, 1999). The prevalence of PMPS-like symptoms in women who had not had surgery for breast cancer is surprisingly high and mandates a conservative view on the raw prevalence figures.The surgical procedures used in the region in the period 2003–2008 have remained principally unchanged. Mammographic screening had been introduced 10 years earlier and thus resulted in an increase in the number of women who could have breast-conserving procedures. The sentinel node technique became more widely used in the last part of the period and this has led to an increase in axillary-sparing procedures.Three risk factors for developing PMPS were identified in this study: having undergone breast surgery earlier, tumour located in the upper lateral quarter and young age. Earlier surgery in the same breast appears to be a logical risk factor for PMPS. Regarding the location of the tumour, previous studies have not evaluated this variable as a risk factor. With tumours in close relation to the axilla, there is a higher risk of damaging nerves in the area that may increase the risk of subsequent chronic pain. Young age has also been reported to be a predisposing factor to PMPS in other studies (Tasmuth et al, 1995; Smith et al, 1999; Poleshuck et al, 2006). It has previously been suggested that this may be caused by the more aggressive character of disease in this group of patients, requiring more invasive surgical procedures and chemotherapy (Kroman et al, 2000; Colleoni et al, 2002). However, in this study, these factors are included in the multiple regression analysis, and this indicates that other factors may account for the fact that PMPS is seen more often among young patients.Two possible risk factors, which have been considered previously, are the sectioning of the intercostobrachial nerve (Abdullah et al, 1998; Torresan et al, 2003) and axillary dissection (Vecht, 1990; Maunsell et al, 1993; Hack et al, 1999; Kakuda et al, 1999; Johansen et al, 2000). However, one study has shown the opposite result, with postmastectomy pain occurring without damage to the intercostobrachial nerve and in women without axillary dissection (Carpenter et al, 1999). In this study, it was not possible to obtain information about the sectioning or preservation of the intercostobrachial nerve among the patients included. Axillary dissection came out as a risk factor in univariate analysis, but this effect could not be reproduced in the multivariate analysis.We did not find any significant difference in the description of pain between breast cancer patients and the reference group. However, the location of the pain did differ between the two groups, with the majority of the breast cancer patients having pain in the shoulder, in the area of the scar and in more than one location. These findings are in agreement with earlier findings (Stevens et al, 1995; Carpenter et al, 1998). When using logistic regression analysis, we found that pain located in the mamma was seen approximately four times less frequent in the group of women who had undergone lumpectomy compared with the women who had undergone mastectomy. Sentinel node biopsy and axillary dissection was not associated with a significant effect on this pain location. Neither the type of breast surgery nor the type of axillary intervention had a significant impact on the frequency of pain located in the mamma. A similar pattern was observed for radiation therapy. An increase in pain located in the breast and especially pain located in the shoulder and arm was seen, when the radiation field included supraclavicular glands and the axillary region. A recent study, including 278 breast cancer patients, has addressed the impact of different types of pain on the degree of disability and distress (Kudel et al, 2007). Three types of pain were studied: phantom breast pain, scar pain and other mastectomy-related pain. In this study, ‘other mastectomy-related pain’ was found to be the strongest predictor of disability and distress. Demographic and surgical factors were not consistent predictors of pain or function.For this type of study, some methodological limitations must be considered. The study group consisted of women who had undergone treatment for breast cancer at one centre, a teaching hospital, and all the women in the study group were Caucasian, that is, we do not know if the figures can be generalised to other settings. Further, it is possible that the percentage of women who did not have PMPS or PMPS-like symptoms was higher in the group of non-responders than in the group of responders. As the response rates in the study group and in the control group were 85 and 73%, respectively, the prevalence of PMPS and the odds ratio for developing PMPS may have been overestimated. There are no specific questionnaires for identifying and evaluating pain after surgery in breast cancer patients, and the questionnaires developed for estimating neuropathic pain components (Bennett, 2001; Krause and Backonja, 2003; Bouhassira et al, 2005; Freynhagen et al, 2006; Portenoy, 2006) were considered not to be suited for the present purpose. Thus, we chose to make a questionnaire specifically for this study and we have no data to support its validity. Women with possible reoccurrence of cancer were included in the study and this is a potential bias of the results. The tumour itself may have caused pain and may thus have caused a higher prevalence of pain in the breast cancer group. Finally, our study comprised 219 patients, and the analysis of the risk factors may have been hampered by the low number of patients.Chronic pain after surgery has been reported to develop in 5–60% of patients after operations such as thoracotomy, hip arthroplasty, hysterectomy, thoracotomy and inguinal hernia repair (Perttunen et al, 1999; Aasvang and Kehlet, 2005; Nikolajsen et al, 2006; Maguire et al, 2006; Pluijms et al, 2006; Kehlet et al, 2006; Brandsborg et al, 2007). For hysterectomy, it was found that about 14% had pelvic pain more than 2 days a week and risk factors were preoperative pelvic pain, pain as the main indicator for surgery and pain problems elsewhere (Brandsborg et al, 2007). Pain after total hip arthroplasty, which limited daily living to a moderate to very severe degree, occurred with a frequency of about 12%, and the risk factors were intensity of early postoperative pain and pain complaints from other areas of the body (Nikolajsen et al, 2006). Prevalence of post-thoracotomy pain was observed to be 21%, when evaluated 6–7 years after surgery, and risk factors were age, consultant and time since surgery (Maguire et al, 2006).Only 22% of the breast cancer patients reported that the pain had an impact on the daily life and use of analgesics was low. These findings suggest that the severity of PMPS, in general, is moderate, which is in agreement with earlier studies (de Vries et al, 1994). 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Curr Med Res Opin\n22(8): 1555–156516870080\n35. Rosso R, Scelsi M, Carnevali L (2000) Granular cell neuroma. Arch Pathol Lab Med\n124: 709–71110782152\n36. Schijven MP, Vingerhoets AJ, Rutten HJ, Nieuwenhuijzen GA, Roumen RM, van Bussel ME, Voogd AC (2003) Comparison of morbidity between axillary lymph node dissection and sentinel node biopsy. Eur J Surg Oncol\n29(4): 341–35012711287\n37. Shons AR, Cox CE (2001) Breast cancer: advances in surgical management. Plast Reconstr Surg\n107(2): 541–54911214073\n38. Smith WCS, Bourne D, Squair J, Phillips DP, Chambers WA (1999) A retrospective cohort study of post mastectomy pain syndrome. Pain\n83: 91–9510506676\n39. Stevens PE, Dibble SL, Miaskowski C (1995) Prevalence, characteristics, and impact of postmastectomy pain syndrome: an investigation of women's experiences. Pain\n61(1): 61–687644250\n40. Tasmuth T, Blomqvist C, Kalso E (1999) Chronic post-treatment symptoms in patients with breast cancer operated in different surgical units. Eur Journal Surg Oncol\n25: 38–4310188853\n41. Tasmuth T, Kataja M, Blomquist C, von Smitten K, Kalso E (1997) Treatment-related factors predisposing to chronic pain in patients with breast cancer. Acta Oncologica\n36(6): 625–6309408154\n42. Tasmuth T, von Smitten K, Hietanen P, Mataja M, Kalso E (1995) Pain and other symptoms after different treatment modalities of breast cancer. Annals Oncol\n6: 453–459\n43. Tasmuth T, von Smitten K, Kalso E (1996) Pain and other symptoms during the first year after radical and conservative surgery for breast cancer. Br J Cancer\n74: 2024–20318980408\n44. Torresan RZ, Cabello C, Conde DM, Brenelli HB (2003) Impact of the preservation of the intercostobrachial nerve in axillary lymphadenectomy due to breast cancer. The Breast J\n9(5): 389–39212968959\n45. Vecht CJ (1990) Arm pain in the patient with breast cancer. J Pain and Symptom Manage\n5(2): 109–1172348086\n46. Wallace MS, Wallace AM, Lee J, Dobke MK (1996) Pain after breast surgery: a survey of 282 women. Pain\n66: 195–2058880841\n47. Wong L (2001) Intercostal neuromas: a treatable cause of postoperative breast surgery pain. Ann Plast Surg\n46: 481–48411352419"
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+    "id": "PMC2527829",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527829\nAUTHORS: K Ota, H Fujimori, M Ueda, S Shiniriki, M Kudo, H Jono, Y Fukuyoshi, Y Yamamoto, H Sugiuchi, H Iwase, M Shinohara, Y Ando\n\nABSTRACT:\nThe aim of this study was to evaluate serum midkine (S-MK) concentrations as a prognostic tumour marker in oral squamous cell carcinoma (OSCC). We measured S-MK concentrations in patients with OSCC and healthy volunteers. In addition, we performed real-time quantitative reverse transcription–PCR analysis and immunohistochemistry with fresh tumour samples. To determine whether S-MK concentrations have prognostic value, we performed survival analyses with clinical information by using the log-rank test. Serum midkine concentrations were significantly higher in patients with OSCC than in healthy controls (P<0.001). Serum midkine concentrations were also significantly increased in early-stage OSCC compared with those of healthy individuals (P<0.001). In addition, immunohistochemistry allowed identification of overexpressed MK protein in OSCC tissues. MK mRNA showed higher expression in OSCC samples compared with normal mucosal samples. Patients in high S-MK groups showed a significantly lower 5-year survival rate compared with patients in low S-MK groups (P<0.05). The increased S-MK concentrations in early-stage OSCC were strongly associated with poor survival. Serum midkine concentrations may thus be a useful marker not only for cancer screening but also for predicting prognosis of OSCC patients.\n\nBODY:\nOral cancer is one of the common types of human cancer (Petersen, 2003; Jemal et al, 2006). The survival rate for patients with oral cancer has not yet improved, despite better diagnostic techniques and innovations in treatments. Improved survival of patients with oral cancer requires better techniques for the prediction of prognosis. Oral squamous cell carcinoma (OSCC), the most common of several types of oral cancers, does not have a good prognosis. Several researchers have studied the usefulness of tumour-associated antigens for primary diagnosis of OSCC, but no tumour markers have given a precise prediction of prognosis (Tsutsui et al, 1993; Krimmel et al, 1998).Midkine (MK), a heparin-binding growth factor, is expressed intensely during the midgestation period and its expression becomes generally weak in adults (Kadomatsu et al, 1988; Muramatsu, 2002). Midkine has various biological activities such as neuronal survival, tissue repair, and carcinogenesis. Midkine, which has neuroprotective activity and neurite extension (Michikawa et al, 1993; Owada et al, 1999), expresses strongly in cerebral infarct (Yoshida et al, 1995) and Alzheimer's disease (Yasuhara et al, 1993; Salama et al, 2005). Midkine promotes the migration of inflammatory cells, specifically macrophages and neutrophils (Takada et al, 1997; Horiba et al, 2000). With these activities, MK expression increases at intraperitoneal adhesions after surgery (Inoh et al, 2004) and cardiac remodelling after myocardial infarction (Obama et al, 1998; Horiba et al, 2006). In addition, MK plays an important role in carcinogenesis. Midkine expression in various malignant tumours is higher than that in normal tissues (Garver et al, 1993, 1994; Konishi et al, 1999; Ye et al, 1999). Midkine protein expression was reported to be correlated with poor prognosis in patients with neuroblastomas (Nakagawara et al, 1995), astrocytomas (Mishima et al, 1997), pancreatic head carcinomas (Maeda et al, 2007), or gastrointestinal stromal tumours (Kaifi et al, 2007). In addition, because MK protein is a plasma-secreted protein, MK levels in blood may increase in patients with malignant diseases. Enzyme-linked immunoassay (EIA) has allowed measurement of MK levels in blood, and increased blood MK levels have been reported in patients with malignant tumours, including hepatocellular carcinoma (Muramatsu et al, 1996), gastric carcinoma, and lung carcinoma (Ikematsu et al, 2000). These reports suggest that MK in blood may have the potential to become a very useful tumour marker. Recent studies have found increased preoperative S-MK concentrations in patients with oesophageal squamous cell carcinoma (Shimada et al, 2003). In addition, higher plasma MK concentrations in patients with neuroblastoma were strongly correlated with poor survival (Ikematsu et al, 2003). Midkine levels in blood may thus become a useful tumour marker for predicting prognosis of cancer patients. However, no studies have focused on the correlation between S-MK expression and OSCC.In this study, we evaluated clinicopathological data and analysed S-MK concentrations in 60 patients with OSCC and 134 healthy volunteers. We used a novel automated fluorescent-enzyme immunoassay (FEIA) system for this analysis. In addition, we compared the expression of MK protein in OSCC tissues and normal mucosal tissues by means of immunohistochemistry and real-time polymerase chain reaction (PCR).Materials and methodsPatients and healthy volunteersA total of 60 patients with primary OSCC who were treated at the Department of Oral and Maxillofacial Surgery (Kumamoto University Hospital), between 1999 and 2004 were studied. We excluded patients who presented with distant metastases and all patients who underwent radical treatments. Fifty-three patients had surgery with more than 10 mm resection margins and no microscopic residual tumour, and of those, 36 patients underwent preoperative chemoradiotherapy. Seven patients underwent radical chemoradiotherapy and we confirmed no residual tumour and no local recurrence. Of the 60 OSCC patients, 37 were males and 23 were females, with a mean age of 66.8±11.4 years (median 67; range 27–92). All patients were staged according to the 1997 UICC TNM Classification of Malignant Tumors (Sobin and Wittekind, 2002). Clinicopathological data, including age, sex, blood data (leukocyte count and platelet count), tumour size, nodal status (N factor; cervical lymph node metastasis), degree of differentiation of OSCC, and prognosis, were obtained from patient files. Serum samples were collected from all patients; biopsy tissue specimens were obtained from 52 patients before treatment.We also collected samples from healthy volunteers who did not have any disorders, such as liver dysfunction and rheumatoid arthritis, after they underwent medical check-ups at Kumamoto University Hospital. In total, we evaluated 134 serum samples and 27 normal oral mucosal specimens, which we excised surgically when we extracted teeth. Of the 134 healthy volunteers, 73 were males and 61 were females, with a mean age of 63.8±11.0 years (median 65; range 20–92). Twenty-seven healthy volunteers (20.1%) and 16 OSCC patients (26.7%) had a smoking habit. Between OSCC patients and healthy volunteers, the smoking habit nearly matched. All subjects gave their informed consent.All serum samples were collected, by means of venipuncture, from patients and volunteers after they gave informed consent. Venous blood was allowed to clot for 30 min at room temperature and was then centrifuged for 10 min at 3000 r.p.m. Serum samples were collected from OSCC patients before treatment. Serum samples and biopsy specimens were stored at –80°C until MK assays and real-time PCR. The remaining tumour materials were fixed in 10% formalin for immunohistochemistry before processing.EIA for MKThe EIA MK assay is a two-site immunoenzymometric assay, requiring 50 μl of serum sample, which was performed automatically using an immunoassay analyser, AIA-600II (Tosoh Corporation, Tokyo, Japan). Midkine in a sample was simultaneously reacted with mouse anti-midkine monoclonal antibody (SC-2) immobilised on magnetisable beads and with alkaline phosphatase-labelled mouse anti-midkine monoclonal antibody (SC-4) to form a sandwich structure. After 10 min of incubation at 37°C, the beads were washed to remove unbound materials. A fluorogenic substrate, 4-methylumbelliferyl phosphate, was added for the enzyme-substrate reaction at 37°C for 5 min. The rate of fluorescence of converted 4-methylumbelliferone was directly proportional to the MK concentration in the samples.RNA isolation and real-time PCRTotal RNA was extracted by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and treated with the RNase-Free DNase Set (Qiagen) according to the manufacturer's instructions. Total RNA was quantified with a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) spectrophotometer and software program. Total RNA (0.5 μg) in samples was reverse transcribed to complementary DNA (cDNA) by using the ExScript RT reagent (Takara Bio Inc., Otsu, Japan), according to the manufacturer's protocol. All PCR reactions were performed by using the LightCycler 480 System (Roche Diagnostics, Basel, Switzerland) with a LightCycler 480 SYBR Green I Master kit (Roche Diagnostics). A reaction mixture was added to 2.0 μl of cDNA for each sample. The reaction mixture contained the following components: 6.0 μl of water, 1 μl of forward primer (0.5 μM), 1 μl of reverse primer (0.5 μM), and 10 μl of 2 × Master mix. The primers used for real-time PCR were as follows: MK forward 5′-AGATGCAGCACCGAGGCT-3′, MK reverse 5′-CTTTCTTTTTGGCGACCG-3′; β2-microgloblin (β2M) forward 5′-CGGGCATTCCTGAAGCTGA-3′, and β2M reverse 5′-GGATGGATGAAACCCAGACACATAG-3′.The β2M gene was chosen for normalisation of data. Each reaction was performed under the following conditions: initialisation for 10 s at 95°C and then 45 cycles of amplification, with 5 s at 95°C for denaturation and 20 s at 60°C for annealing and elongation. After amplification, the temperature was slowly raised to above the melting temperature of the PCR product to measure fluorescence and thereby to determine the melting curve. In addition, to ensure RNA quality, several preparations were subjected to analysis of expression using the Hitachi SV1210 microchip electrophoresis system (Hitachi Electronics Engineering Company, Tokyo, Japan). A standard curve was plotted for each primer probe established by using serial dilution of pooled cDNA from tissues. All standards and samples were analysed in triplicate.ImmunohistochemistryTo detect MK in OSCC samples, we performed immunohistochemical analysis with two monoclonal MK antibodies: IP-10, with an epitope in the N-terminal half of the MK fragment, and IP-14, reacting with the C-terminal half of the MK fragment. The primary antibodies IP-10 and IP-14 were generated by Cell Signals Inc. (Yokohama, Japan). Paraffin-embedded 4-μm-thick sections were prepared, deparaffinised in xylene, and rehydrated in graded alcohols. Endogenous peroxidase activity was blocked by immersing sections in 0.3% hydrogen peroxide in methanol for 30 min, and antigen retrieval was performed by a 15-min microwave pretreatment in citrate buffer (pH 6.0, 0.01 mol l−1). After being incubated with Protein Block Serum-Free (Dako, Glostrup, Denmark), sections were incubated overnight at 4°C with mouse monoclonal antibodies (IP-10 at 1 : 25; IP-14 at 1 : 100). After incubation with antimouse labelled polymer (EnVision+ System HPR; Dako) for 30 min at room temperature, 3,3′-diaminobenzidine was used as the chromogen. Sections were immunohistochemically stained and were then counterstained with hematoxylin to enhance nuclear detection.Statistical analysisThe normality of the data was first assessed using the Shapiro–Wilks test. Data were then evaluated using Student's t-test and an analysis of variance (normal distributed data) or by the Kruskall–Wallis test (non-normal data). Regression analysis was done by simple regression on S-MK concentrations to leukocyte or platelet. To determine the cutoff point of S-MK concentrations, receiver-operating characteristic (ROC) curves were constructed by calculating the sensitivities and specificities for cutoff values. The optimal cutoff values were selected on the basis of the extreme upper left points of the ROC curves. Survival curves were plotted by using the Kaplan–Meier method and analysed with the log-rank test for univariate analysis. All analyses were performed with the JMP (version 5.0.1; SAS Institute Japan, Tokyo, Japan). P-values of less than 0.05 were regarded as statistically significant.ResultsS-MK concentration and its correlation with prognosis in OSCCWe determined S-MK concentrations in samples from 60 OSCC patients and 134 healthy volunteers. The mean (±s.d.) S-MK concentration was 885.9±465.0 pg ml−1 for the OSCC patients and 419.1±97.9 pg ml−1 for the healthy subjects. Serum midkine concentrations for the OSCC patients were significantly higher than those for healthy volunteers (Figure 1). To examine the usefulness of this marker protein, a cutoff point was determined for each set of values, by using a ROC curve, under the condition of specificity equal to 99.3%. With a cutoff value of 650, 66.7% of the OSCC patients had S-MK values that were positive predictive markers. As Table 1 shows, S-MK concentrations in patients and healthy subjects older than 60 years were significantly higher compared with those in patients and healthy subjects younger than 60 years, respectively. We compared S-MK concentrations in healthy volunteers and OSCC patients under 60 and over 60 years old. Serum midkine concentrations of OSCC patients were significantly higher than those in healthy volunteers over 60 years old (965.8±505.5 and 456.3±119.2 pg ml−1, respectively). Moreover, in the analysis of both groups under the age of 60 years old, the concentrations in OSCC patients were significantly higher than those in healthy volunteers (645.9±160.8 and 345.3±93.6 pg ml−1, respectively). We concluded from these analyses that, independently from the ages, S-MK concentrations in OSCC patients were significantly higher than those in age-matched healthy volunteers. We also studied S-MK concentrations in relation to clinicopathological parameters (sex, age, smoking habit, clinical stage, tumour size, cervical lymph node metastasis, degree of differentiation of OSCC, and prognosis) in OSCC patients (Tables 1 and 2). However, we found no correlation between S-MK and sex, tumour size, cervical lymph node metastasis, or degree of differentiation of OSCC. Although S-MK concentrations showed no increasing tendency related to clinical stage, concentrations were significantly elevated in all stages in OSCC compared with those in healthy volunteers (stage I, III, IV; P<0.001, stage II; P<0.01) (Figure 2). We measured leukocyte and platelet counts to examine the influence of S-MK concentrations. With simple regression analysis, a relationship was not found between S-MK concentrations and the numbers of leukocytes or platelets (leukocytes r=0.03 P=0.80; platelets r=0.02 P=0.90). We also measured the MK concentration in plasma in 60 OSCC patients and 91 healthy volunteers. Plasma MK (P-MK) concentrations in OSCC patients and healthy volunteers were 783.6±286.1 and 404.2±89.3 pg ml−1, respectively. Oral squamous cell carcinoma patients had significantly higher P-MK concentrations than did healthy volunteers (P<0.0001).In our investigation of the relationship between S-MK concentrations and prognosis, we divided OSCC S-MK concentrations into two groups: higher than or equal to 650 pg ml−1 (n=40) or lower than 650 pg ml−1 (n=20). The lower S-MK concentration group had a significantly greater 5-year survival rate than did the higher S-MK concentration group (82.9 vs 56.6%; P<0.05) (Figure 3). The median survival time of OSCC patients with high S-MK groups was 62 months, but the survival rate in low S-MK groups was not below 50%, whereas the average observation period after radical treatment was 69.2 months. We observed no significant relationship between 5-year survival rate and age. However, as Table 2 shows, clinical stage, tumour size, and cervical lymph node metastasis were significantly related to 5-year survival rate.MK mRNA levels in OSCCTo examine the relative expression of the MK gene in OSCC samples, we used real-time PCR to determine MK mRNA levels in 60 samples from OSCC patients and 28 samples from healthy volunteers. Midkine mRNA levels were significantly higher in OSCC tissues than in normal mucosal tissues (0.28 vs 0.15 P<0.05) (Figure 4). However, MK mRNA expression in each OSCC tissue specimen was not significantly associated with S-MK concentration (data not shown).Immunohistochemical staining of MK in OSCCImmunoreactive MK was localised in cancer cell cytoplasm. Out of 52 OSCC samples evaluated with IP-10 antibody, 45 were immunoreactive; with IP-14 antibody, 43 samples also showed a positive reaction. However, differences in reactivity – chromatic strength and localisation of staining – were noted with the IP-10 and IP-14 antibodies. Recognition of tumour cells by IP-10 was greater, and only IP-14 was immunoreactive with glandular duct (Figure 5).DiscussionOur study produced two important findings. First, even though S-MK concentrations were not related to clinical stage, tumour size, and cervical lymph node metastasis, S-MK concentration offers promise as a novel tumour marker of prognosis in OSCC. Serum midkine concentrations will allow us to predict prognosis because, in the presence of highly elevated concentrations, we would use a larger surgically margin when removing a tumour and would perform chemotherapy and/or radiotherapy before or after the operation with better results. Midkine has been well documented to promote various activities related to oncogenesis and tumour progression, including cell migration (Takada et al, 1997), angiogenic functions (Choudhuri et al, 1997), mitogenesis (Muramatsu and Muramatsu, 1991), and antiapoptosis (Owada et al, 1999). Midkine can also give cancerous cells a growth advantage through antiapoptotic activity. Qi et al (2000) observed that MK protein rescued G401 cells, a Wilms’ tumour cell line, from cisplatin-induced apoptosis by upregulation of Bcl-2. Mirkin et al (2005) reported that increased MK expression exerted a significant cytoprotective effect against doxorubicin in drug-sensitive cells. They suggested that MK indirectly mediates acquired drug resistance to protect neighbouring drug-sensitive cells and contributes to the development of resistance to chemotherapeutics. In 40 patients who underwent an operation and chemotherapy, S-MK concentrations tended to be higher in the group with recurrence and/or metastasis than in the group without recurrence or metastasis (Figure 6). We confirmed the belief that MK directly or indirectly aids survival of cancer cells through antiapoptosis and provides cytoprotection against chemotherapy and radiotherapy. Takei et al (2006) reported that a combined therapy involving MK small interfering RNA and Paclitaxel significantly enhanced anticancer activity or maintained the effective anticancer activity of Paclitaxel. To inhibit the secretion of MK may be a novel therapy against drug-resistant cancers. In addition, MK protein expression in OSCC cases was reported to be significantly correlated with the expression of vascular endothelial growth factor (VEGF) (Ruan et al, 2007), and S-MK concentration was correlated with serum levels of VEGF-C (Krzystek-Korpacka et al, 2007). Expression of VEGF was also significantly correlated with prognosis in OSCC (Uehara et al, 2004). In these previous results, MK may be related to a prognosis through the activation of VEGF. However, it was reported that MK downregulated VEGF-A-induced neovascularisation and vascular permeability in a recent study (van der Horst et al, 2008). The involvement of MK in tumour angiogenesis warrants further investigation and should include a study of VEGF.Second, we believe that S-MK concentration can become a tool for screening of OSCC. Serum midkine concentrations in patients with early-stage OSCC were significantly higher than those in healthy individuals. This result suggests that S-MK concentration may assist in the early detection of not only OSCC but also other tumours. We measured P-MK concentrations to investigate whether MK leaks from platelets in blood coagulation and an anticoagulant have an influence on the expression of MK. In P-MK concentrations, those values were more elevated in OSCC patients than in healthy volunteers as did in S-MK concentrations. In addition, P-MK concentrations in OSCC patients in the early stage were significantly higher than those in healthy individuals. Platelets and leukocytes in OSCC patients did not relate to the expression of MK. These results suggest that P-MK and S-MK concentrations are useful for early diagnosis of oral cancer. However, concerning the collection of plasma samples, we could not collect those for a long period. As the collected samples were all within 4 years after the surgery, we could not calculate the 5-year survival rate. In reports on other tumours, such as neuroblastoma (Ikematsu et al, 2003), oesophageal cancer (Shimada et al, 2003), and gastric cancer (Obata et al, 2005), serum or plasma MK concentrations were significantly elevated. Because MK is a plasma protein, serum or plasma MK concentrations can be elevated in various types of cancer. As we demonstrated here, an automated FEIA system may be useful in screening for cancer in a large number of samples in a short time.In both OSCC patients and healthy subjects, S-MK concentrations older than 60 years were significantly higher compared with those younger than 60 years. These analyses suggest that ageing may influence S-MK concentrations. On the basis of the past reports about MK expression in ageing-related diseases, it is natural to consider that expression of MK is elevated in the presymptomatic stages of diseases, such as asymptomatic cerebral infarction, asymptomatic ischemic heart diseases, and a pathological prestage of Alzheimer's disease.Figures 4 and 5 demonstrated, by means of immunohistochemistry and mRNA MK analysis, that MK protein was overexpressed in OSCC tissues compared with healthy tissue from volunteers. However, MK mRNA expression in OSCC tissue specimens was not significantly associated with S-MK concentrations. Serum midkine concentrations were previously reported to be associated with MK protein expression in oesophageal cancer cells (Shimada et al, 2003), and the 5-year survival rate of a group with high MK expression in OSCC tissue was lower than that of a group with low MK expression (Ruan et al, 2007). These results suggest that MK overexpression in OSCC tissues may promote high concentrations of S-MK. Expression of MK may be localised in cancer tissues, because RNA was extracted from some cancer tissues. However, we found no relationship between tumour size and S-MK concentrations and no correlation between MK mRNA expression and 5-year survival rates (data not shown).Expression of a truncated form of MK (t-MK) mRNA, which lacks exon 3 encoding the N terminus, has been well documented in various tumours, including colon (Miyashiro et al, 1996), breast (Miyashiro et al, 1997), gastric (Aridome et al, 1998), and liver and kidney (Tao et al, 2007). Therefore, we immunostained OSCC tissues with IP-14 antibody, which had an epitope in the C-terminus, and IP-10 antibody, which had an epitope in the N-terminus. One might expect that IP-14, which recognised both the full-length and truncated forms of MK, would show a stronger reaction in carcinoma than IP-10, which did not cross-react with t-MK. However, our results did not support this expectation. IP-14 reactivity was weaker than IP-10 reactivity (Figure 5). Our result agrees with the report of Nobata and colleagues (Nobata et al, 2005) that the C-terminus can easily be affected by protease and that t-MK antibody has a low affinity for the C-terminus. Moreover, we first believed that IP-10 would strongly react with MK functioning in protection of the N-terminal part of the molecule from proteolytic degradation (Matsuda et al, 1996). However, glandular ducts stained only with IP-14 antibody. This finding indicates that t-MK may be expressed in normal salivary glands, although previous studies reported that t-MK was not detected in noncancerous tissues. Studies to clarify the localisation of t-MK are in progress. On the basis of these results, we analysed S-MK concentrations by using SC2 and SC4 antibodies, which had epitopes in the N-terminus, as does the IP-10 antibody. Even though these antibodies did not react with t-MK, they could detect full-length MK, and the sandwich ELISA performed with these two antibodies was specific and useful for cancer screening and predicting the prognosis of OSCC patients.In conclusion, S-MK concentrations in OSCC patients were associated with prognosis (5-year survival) but not with conventional prognostic factors such as clinical stage, tumour size, and cervical lymph node metastasis. Midkine expression in blood and cancer tissues is indicative of a strong relationship with malignant potential, and high MK expression suggests a bad prognosis. These studies may lead to trials to inhibit MK expression by using MK antibody and small interfering RNA. The study of MK may elucidate problems of anticancer drug resistance in various cancers and contribute to improvement in prognosis.\n\nREFERENCES:\n1. Aridome K, Takao S, Kaname T, Kadomatsu K, Natsugoe S, Kijima F, Aikou T, Muramatsu T (1998) Truncated midkine as a marker of diagnosis and detection of nodal metastases in gastrointestinal carcinomas. Br J Cancer\n78: 472–4779716029\n2. Choudhuri R, Zhang HT, Donnini S, Ziche M, Bicknell R (1997) An angiogenic role for the neurokines midkine and pleiotrophin in tumorigenesis. Cancer Res\n57: 1814–18199135027\n3. Garver Jr RI, Chan CS, Milner PG (1993) Reciprocal expression of pleiotrophin and midkine in normal vs malignant lung tissues. Am J Respir Cell Mol Biol\n9: 463–4668217186\n4. Garver Jr RI, Radford DM, Donis-Keller H, Wick MR, Milner PG (1994) Midkine and pleiotrophin expression in normal and malignant breast tissue. Cancer\n74: 1584–15907520350\n5. Horiba M, Kadomatsu K, Nakamura E, Muramatsu H, Ikematsu S, Sakuma S, Hayashi K, Yuzawa Y, Matsuo S, Kuzuya M, Kaname T, Hirai M, Saito H, Muramatsu T (2000) Neointima formation in a restenosis model is suppressed in midkine-deficient mice. J Clin Invest\n105: 489–49510683378\n6. Horiba M, Kadomatsu K, Yasui K, Lee JK, Takenaka H, Sumida A, Kamiya K, Chen S, Sakuma S, Muramatsu T, Kodama I (2006) Midkine plays a protective role against cardiac ischemia/reperfusion injury through a reduction of apoptotic reaction. Circulation\n114: 1713–172017015789\n7. Ikematsu S, Nakagawara A, Nakamura Y, Sakuma S, Wakai K, Muramatsu T, Kadomatsu K (2003) Correlation of elevated level of blood midkine with poor prognostic factors of human neuroblastomas. Br J Cancer\n88: 1522–152612771916\n8. Ikematsu S, Yano A, Aridome K, Kikuchi M, Kumai H, Nagano H, Okamoto K, Oda M, Sakuma S, Aikou T, Muramatsu H, Kadomatsu K, Muramatsu T (2000) Serum midkine levels are increased in patients with various types of carcinomas. Br J Cancer\n83: 701–70610952771\n9. Inoh K, Muramatsu H, Ochiai K, Torii S, Muramatsu T (2004) Midkine, a heparin-binding cytokine, plays key roles in intraperitoneal adhesions. Biochem Biophys Res Commun\n317: 108–11315047154\n10. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, Thun MJ (2006) Cancer statistics, 2006. CA Cancer J Clin\n56: 106–13016514137\n11. Kadomatsu K, Tomomura M, Muramatsu T (1988) cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun\n151: 1312–13183355557\n12. Kaifi JT, Fiegel HC, Rafnsdottir SL, Aridome K, Schurr PG, Reichelt U, Wachowiak R, Kleinhans H, Yekebas EF, Mann O, Ichihara-Tanaka K, Muramatsu T, Kluth D, Strate T, Izbicki JR (2007) Midkine as a prognostic marker for gastrointestinal stromal tumors. J Cancer Res Clin Oncol\n133: 431–43517221186\n13. Konishi N, Nakamura M, Nakaoka S, Hiasa Y, Cho M, Uemura H, Hirao Y, Muramatsu T, Kadomatsu K (1999) Immunohistochemical analysis of midkine expression in human prostate carcinoma. Oncology\n57: 253–25710545795\n14. Krimmel M, Hoffmann J, Krimmel C, Cornelius CP, Schwenzer N (1998) Relevance of SCC-Ag, CEA, CA 19.9 and CA 125 for diagnosis and follow-up in oral cancer. J Craniomaxillofac Surg\n26: 243–2489777503\n15. Krzystek-Korpacka M, Matusiewicz M, Diakowska D, Grabowski K, Blachut K, Kustrzeba-Wojcicka I, Banas T (2007) Serum midkine depends on lymph node involvement and correlates with circulating VEGF-C in oesophageal squamous cell carcinoma. Biomarkers\n12(4): 403–41317564845\n16. Maeda S, Shinchi H, Kurahara H, Mataki Y, Noma H, Maemura K, Aridome K, Yokomine T, Natsugoe S, Aikou T, Takao S (2007) Clinical significance of midkine expression in pancreatic head carcinoma. Br J Cancer\n97: 405–41117622248\n17. Matsuda Y, Talukder AH, Ishihara M, Hara S, Yoshida K, Muramatsu T, Kaneda N (1996) Limited proteolysis by chymotrypsin of midkine and inhibition by heparin binding. Biochem Biophys Res Commun\n228: 176–1818912655\n18. Michikawa M, Kikuchi S, Muramatsu H, Muramatsu T, Kim SU (1993) Retinoic acid responsive gene product, midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture. J Neurosci Res\n35: 530–5398377224\n19. Mirkin BL, Clark S, Zheng X, Chu F, White BD, Greene M, Rebbaa A (2005) Identification of midkine as a mediator for intercellular transfer of drug resistance. Oncogene\n24: 4965–497415897897\n20. Mishima K, Asai A, Kadomatsu K, Ino Y, Nomura K, Narita Y, Muramatsu T, Kirino T (1997) Increased expression of midkine during the progression of human astrocytomas. Neurosci Lett\n233: 29–329324232\n21. Miyashiro I, Kaname T, Nakayama T, Nakamori S, Yagyu T, Monden T, Kikkawa N, Nishisho I, Muramatsu T, Monden M, Akiyama T (1996) Expression of truncated midkine in human colorectal cancers. Cancer Lett\n106: 287–2918844985\n22. Miyashiro I, Kaname T, Shin E, Wakasugi E, Monden T, Takatsuka Y, Kikkawa N, Muramatsu T, Monden M, Akiyama T (1997) Midkine expression in human breast cancers: expression of truncated form. Breast Cancer Res Treat\n43: 1–69065593\n23. Muramatsu H, Muramatsu T (1991) Purification of recombinant midkine and examination of its biological activities: functional comparison of new heparin binding factors. Biochem Biophys Res Commun\n177: 652–6582049087\n24. Muramatsu H, Song XJ, Koide N, Hada H, Tsuji T, Kadomatsu K, Inui T, Kimura T, Sakakibara S, Muramatsu T (1996) Enzyme-linked immunoassay for midkine, and its application to evaluation of midkine levels in developing mouse brain and sera from patients with hepatocellular carcinomas. J Biochem\n119: 1171–11758827454\n25. Muramatsu T (2002) Midkine and pleiotrophin: two related proteins involved in development, survival, inflammation and tumorigenesis. J Biochem\n132: 359–37112204104\n26. Nakagawara A, Milbrandt J, Muramatsu T, Deuel TF, Zhao H, Cnaan A, Brodeur GM (1995) Differential expression of pleiotrophin and midkine in advanced neuroblastomas. Cancer Res\n55: 1792–17977712489\n27. Nobata S, Shinozawa T, Sakanishi A (2005) Truncated midkine induces transformation of cultured cells and short latency of tumorigenesis in nude mice. Cancer Lett\n219: 83–8915694667\n28. Obama H, Biro S, Tashiro T, Tsutsui J, Ozawa M, Yoshida H, Tanaka H, Muramatsu T (1998) Myocardial infarction induces expression of midkine, a heparin-binding growth factor with reparative activity. Anticancer Res\n18: 145–1529568069\n29. Obata Y, Kikuchi S, Lin Y, Yagyu K, Muramatsu T, Kumai H (2005) Serum midkine concentrations and gastric cancer. Cancer Sci\n96: 54–5615649256\n30. Owada K, Sanjo N, Kobayashi T, Mizusawa H, Muramatsu H, Muramatsu T, Michikawa M (1999) Midkine inhibits caspase-dependent apoptosis via the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase in cultured neurons. J Neurochem\n73: 2084–209210537068\n31. Petersen PE (2003) The World Oral Health Report 2003: continuous improvement of oral health in the 21st century – the approach of the WHO Global Oral Health Programme. Community Dent Oral Epidemiol\n31(Suppl 1): 3–2315015736\n32. Qi M, Ikematsu S, Ichihara-Tanaka K, Sakuma S, Muramatsu T, Kadomatsu K (2000) Midkine rescues Wilms’ tumor cells from cisplatin-induced apoptosis: regulation of Bcl-2 expression by Midkine. J Biochem\n127: 269–27710731694\n33. Ruan M, Ji T, Wu Z, Zhou J, Zhang C (2007) Evaluation of expression of midkine in oral squamous cell carcinoma and its correlation with tumour angiogenesis. Int J Oral Maxillofac Surg\n36: 159–16417110085\n34. Salama RH, Muramatsu H, Shimizu E, Hashimoto K, Ohgake S, Watanabe H, Komatsu N, Okamura N, Koike K, Shinoda N, Okada S, Iyo M, Muramatsu T (2005) Increased midkine levels in sera from patients with Alzheimer’s disease. Prog Neuropsychopharmacol Biol Psychiatry\n29: 611–61615866365\n35. Shimada H, Nabeya Y, Tagawa M, Okazumi S, Matsubara H, Kadomatsu K, Muramatsu T, Ikematsu S, Sakuma S, Ochiai T (2003) Preoperative serum midkine concentration is a prognostic marker for esophageal squamous cell carcinoma. Cancer Sci\n94: 628–63212841873\n36. Sobin LH, Wittekind C (2002) TNM Classification of Malignant Tumours (6th ed), Vol. 6th edn. Wiley-Liss: New York\n37. Takada T, Toriyama K, Muramatsu H, Song XJ, Torii S, Muramatsu T (1997) Midkine, a retinoic acid-inducible heparin-binding cytokine in inflammatory responses: chemotactic activity to neutrophils and association with inflammatory synovitis. J Biochem\n122: 453–4589378726\n38. Takei Y, Kadomatsu K, Goto T, Muramatsu T (2006) Combinational antitumor effect of siRNA against midkine and paclitaxel on growth of human prostate cancer xenografts. Cancer\n107: 864–87316832814\n39. Tao P, Xu D, Lin S, Ouyang GL, Chang Y, Chen Q, Yuan Y, Zhuo X, Luo Q, Li J, Li B, Ruan L, Li Q, Li Z (2007) Abnormal expression, highly efficient detection and novel truncations of midkine in human tumors, cancers and cell lines. Cancer Lett\n253: 60–6717379400\n40. Tsutsui J, Kadomatsu K, Matsubara S, Nakagawara A, Hamanoue M, Takao S, Shimazu H, Ohi Y, Muramatsu T (1993) A new family of heparin-binding growth/differentiation factors: increased midkine expression in Wilms’ tumor and other human carcinomas. Cancer Res\n53: 1281–12858383007\n41. Uehara M, Sano K, Ikeda H, Sekine J, Irie A, Yokota T, Tobita T, Ohba S, Inokuchi T (2004) Expression of vascular endothelial growth factor and prognosis of oral squamous cell carcinoma. Oral Oncol\n40: 321–32514747064\n42. van der Horst EH, Frank BT, Chinn L, Coxon A, Li S, Polesso F, Slavin A, Ruefli-Brasse A, Wesche H (2008) The growth factor Midkine antagonizes VEGF signaling in vitro and in vivo. Neoplasia\n10: 340–34718392135\n43. Yasuhara O, Muramatsu H, Kim SU, Muramatsu T, Maruta H, McGeer PL (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer disease. Biochem Biophys Res Commun\n192: 246–2518476427\n44. Ye C, Qi M, Fan QW, Ito K, Akiyama S, Kasai Y, Matsuyama M, Muramatsu T, Kadomatsu K (1999) Expression of midkine in the early stage of carcinogenesis in human colorectal cancer. Br J Cancer\n79: 179–18410408712\n45. Yoshida Y, Goto M, Tsutsui J, Ozawa M, Sato E, Osame M, Muramatsu T (1995) Midkine is present in the early stage of cerebral infarct. Brain Res Dev Brain Res\n85: 25–307781164"
+}

batch_10/PMC2527831.json

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+{
+    "id": "PMC2527831",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527831\nAUTHORS: J F Timms\n\nABSTRACT:\nNo Abstract\n\nBODY:\nProteomic technologies hold great promise in the search for clinically useful protein biomarkers for the early detection, diagnosis and prognosis of cancer and for monitoring response to therapy. Moreover, they have the potential for contributing to the discovery of novel drug targets and for testing the biochemical effects of drugs on cells and tissues and to define cancer-related molecular events. This book, from the Cancer Drug Discovery and Development Series, provides the reader with a current perspective and anticipated uses of proteomic strategies in cancer therapy as well as basic cancer research.The book is divided into four parts, beginning with a detailed background on current proteomic technologies. The chapter describes mass spectrometry-based methods and strategies for studying protein phosphorylation and the quantitation of proteins for expression profiling. Although a technical subject, the authors have not overcomplicated the topic, and the chapter provides the non-specialist reader with an understandable background to the methods and also details some of the technical challenges associated with proteomic analysis. The second part of the book plunges the reader into the topic of cell signalling proteomics, with two very specific chapters on the integration of genomics and proteomics for the study of p53 signalling, and the profiling of tyrosine kinases as pharmacological end points for targeted cancer therapy. The general methods and background to the signalling pathways and tyrosine kinase inhibitors with therapeutic use are well described in both chapters, and work in these areas is well-referenced. However, chapter 2 includes an abundance of experimental data from the authors’ own laboratory, drawing the reader away from the focus of the book, and although chapter 3 promises an interesting section on clinical phospho-proteomics, it fails to deliver.The third part of the book describes clinical applications of proteomics in cancer therapy, although few, if any, proteomic-based tests are actually used in the clinic. This part of the book is perhaps inaccurately entitled ‘Tumour Proteomics’, as it covers an array of topics from oncoproteomics for personalised management, through serum proteomics for cancer detection, to strategies of therapeutic individualisation and target discovery in AML. In chapter 4, the concept of the cancer biomarker is introduced along with the role of proteomics in the discovery of biomarkers for diagnosis, monitoring cancer progression, predicting recurrence, assessing response/efficacy of treatment and selecting patients for given therapies. Several more methodologies and strategies for biomarker discovery are briefly described, although are inadequately referenced. The section is poorly organised and it is not clear how clinically useful such approaches are or will be in the future, particularly for directing personalised therapies. Chapter 5 reads much better, dealing with serum and tissue proteomics and the incorporation of proteomic tests into clinical trials where they are expected to provide useful end points. The introduction describes currently used serum tumour markers, discusses their inadequacies and emphasizes the need for better cancer biomarkers. Several proteomics methods are again described (gel electrophoresis, mass spectrometry, protein arrays, and so on), which would have been better placed in the first section of the book.Chapters 6–8 provide specific examples of the use of proteomics in the study of renal cell carcinoma, lung cancer and acute leukaemia. Again there are informative backgrounds for the non-specialist reader and several interesting sections on tumour antigens, the importance of sample selection and preparation, molecular diagnosis and classification of cancer using proteomics, multiplexed immune-based protein assays and drug target validation using proteomics. Chapter 9 is excellent and deals with practical aspects of tumour biomarker discovery; a must for all new researchers to the field, although would have been better placed earlier in the book. It describes guidelines for the development and introduction of biomarkers to the clinic and has first-rate sections on pre-analytical, analytical and post-analytical aspects of biomarker assessment and the clinical uses of biomarkers in cancer.The final part of the book covers bioinformatics and regulatory aspects of proteomics. Chapter 10 provides a useful overview on efforts to annotate the human proteome, references useful data resources and, importantly, highlights the need and global initiatives for standardising the reporting and storage of proteomic information from different sources. The final chapter deals with regulatory issues in the co-development of drugs and proteomic tests. It includes the role of the Food and Drug Administration (FDA) in regulating the use of proteomics in cancer therapy and covers issues of target characterisation, intended use and indication for use, method and operation standardisation and the importance of study design.Despite the reservations outlined above, the book provides a broad account of the area of cancer proteomics and adequately covers issues relevant to the use of proteomic strategies in clinical research. There are useful reviews of different methodologies, work in different cancer types, on biomarker development and clinical use and on bioinformatics and regulatory aspects of proteomics. The book would be of interest to clinicians and basic cancer researchers alike who are already involved in, or who are considering the use of proteomics in their work. However, the book also reveals that clinical proteomics is still in its infancy, that biomarker research is firmly in the early discovery phase and that there has been little clinical validation of the many recently reported ‘biomarkers’ of cancer. Thus, although proteomics has contributed significantly to our understanding of the molecular biology of cancer, it is a sad fact it has yet to deliver any clinically useful biomarkers.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2527839.json

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+{
+    "id": "PMC2527839",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527839\nAUTHORS: J-L Lee, Y-K Kang, H J Kang, K-H Lee, D Y Zang, B-Y Ryoo, J G Kim, S R Park, W K Kang, D B Shin, M-H Ryu, H M Chang, T-W Kim, J H Baek, Y J Min\n\nABSTRACT:\nThis randomised multicentre phase II study was conducted to investigate the activity and safety of two oral fluoropyrimidines, capecitabine or S-1, in elderly patients with advanced gastric cancer (AGC). Elderly (⩾65 years) chemo-naive patients with AGC were randomly assigned to receive capecitabine 1250 mg m−2 two times daily on days 1–14 every 3 weeks or S-1 40–60 mg two times daily according to body surface area on days 1–28 every 6 weeks. Ninety-six patients were enrolled and 91 patients were randomised to capecitabine (N=46) or S-1 (N=45). Overall response rate, the primary end point, was 27.2% (95% CI, 14.1–40.4, 12 of 44 assessable patients) with capecitabine and 28.9% (95% CI, 15.6–42.1, 13 of 45) with S-1. Median times to progression and overall survival in the capecitabine arm (4.7 and 9.5 months, respectively) were similar to those in the S-1 arm (4.2 and 8.2 months, respectively). The incidence of grade 3–4 granulocytopenia was 6.8% with capecitabine and 4.8% with S-1. Grade 3–4 nonhaematologic toxicities were: asthenia (9.1% with capecitabine vs 7.1% with S-1), anorexia (6.8 vs 9.5%), diarrhoea (2.3 vs 0%), and hand–foot syndrome (6.8 vs 0%). Both capecitabine and S-1 monotherapies were active and tolerable as first-line treatment for elderly patients with AGC.\n\nBODY:\nStomach cancer is the fourth most frequent malignant disease and the second most common cause of cancer-related deaths in the world (Parkin et al, 2005; Ries et al, 2007). Although the number of deaths from gastric cancer has declined over the past decades as a result of screening endoscopy, the elderly are primarily affected by the disease with most gastric cancer-related deaths occurring in patients aged 65 years or older (Ries et al, 2007).Systemic chemotherapy improves the quantity and quality of life in patients with gastric cancer when compared with best supportive care alone (Murad et al, 1993; Pyrhonen et al, 1995; Glimelius et al, 1997). Various chemotherapeutic agents are used for the treatment of advanced gastric cancer (AGC). Some are used as single agents, others as part of combination regimens. 5-Fluorouracil (5-FU) has been the backbone of most regimens for AGC for several decades, and is used most commonly in combination with a platinum with or without an anthracycline or taxane (Kim et al, 1993; Webb et al, 1997; Waters et al, 1999; Vanhoefer et al, 2000; Van Cutsem et al, 2006). However, elderly cancer patients often present with concomitant co-morbidities and age-associated physiologic problems, such as impaired organ function and functional changes that make the selection of optimal treatment difficult (Repetto and Balducci, 2002; Lichtman et al, 2007b). There is also uncertainty about the use of systemic palliative chemotherapy in elderly patients because of under-representation of this age group in clinical trials (Murthy et al, 2002; Lewis et al, 2003; Lichtman et al, 2007a).Capecitabine is an oral fluoropyrimidine carbamate, which is enzymatically converted to 5-FU in several steps following absorption from the gastrointestinal tract. The final step involves the enzyme thymidine phosphorylase, which is found at much higher levels in gastric cancers than in normal tissue, enabling the active drug 5-FU to be generated preferentially at the tumour site (Miwa et al, 1998). The efficacy and safety of the standard 3-weekly intermittent regimen of capecitabine for AGC has been demonstrated in a Korean study including 55 chemo-naive patients (Hong et al, 2004). The overall response rate (ORR) was 34% with a median overall survival (OS) of 9.5 months with a favourable safety profile. More recently, two randomised phase III trials in patients with AGC have been completed. The first multinational study showed that capecitabine/cisplatin was noninferior to 5-FU/cisplatin in terms of progression-free survival (Kang et al, 2006). The second trial (REAL 2), which was performed in the UK and Australia, demonstrated that capecitabine can replace 5-FU and oxaliplatin can replace cisplatin in triple combinations used for the treatment of advanced esophagogastric cancer (Cunningham et al, 2008). Capecitabine has since received approval for use in AGC on the basis of these trials.S-1 is a novel oral fluoropyrimidine consisting of a 5-FU prodrug, tegafur, and the dihydropyrimidine dehydrogenase inhibitor, 5-chloro-2,4-dihydroxypyridine and the orotate phosphoribosyl transferase inhibitor, potassium oxonate, which suppresses the gastrointestinal toxicity of tegafur (Maehara, 2003). In two phase II studies in patients with AGC conducted in Japan, 80 mg m−2 of S-1 daily for 4 weeks followed by a 2-week rest period showed high ORRs of 49% (25 of 51) and 44% (19 of 43), respectively, with median OS of 8.3 and 6.8 months, respectively (Sakata et al, 1998; Koizumi et al, 2000). By virtue of their oral formulations, promising efficacy, and favourable toxicity profiles, capecitabine and S-1 may be particularly attractive for elderly cancer patients.This phase II trial (NCT00278863) was conducted to evaluate the efficacy, safety, and feasibility of capecitabine or S-1 in elderly patients with previously untreated AGC and to assess the relative advantages of these agents in the treatment of AGC.Materials and methodsPatientsElderly patients (aged⩾65 years) with advanced, unresectable, histologically confirmed adenocarcinoma of the stomach or gastroesophageal junction were eligible if they met the following inclusion criteria: ECOG performance status 0–2; measurable disease based on RECIST criteria; no previous chemotherapy except adjuvant chemotherapy completed at least 12 months before enrollment; an estimated life expectancy of more than 3 months; ability for adequate oral intake; adequate bone marrow function, defined as leukocyte count ⩾4000 μl−1, absolute neutrophil count (ANC) ⩾1500 μl−1, haemoglobin ⩾9.0 g dl−1, and platelets ⩾100 000 μl−1; adequate renal and hepatic function, defined as serum creatinine <1.5 mg dl−1, bilirubin <1.5 mg dl−1, and serum transaminase <3 times the upper normal limit (UNL) (<5 times UNL for patients with liver metastases); and written informed consent. Patients were excluded if they had brain metastases, significant gastrointestinal bleeding, a serious co-morbid condition, concomitant use of any drugs, which had a potential interaction with S-1 (flucytosine, allopurinol, warfarin, and phenytoin), or inability to comply with the requirements of the protocol. This study was initially approved by the Institutional Review Board (IRB) of the Asan Medical Center and later by all IRBs responsible for the participating centres.Study design and randomisationThis study was an open-label, multicentre, randomised phase II trial with two treatment arms. Random assignment (1 : 1 ratio) was centralised and performed by the Coordination Center for Clinical Trials on Gastrointestinal Cancer at the Asan Medical Center, Seoul, Korea. Randomisation was stratified by age (⩽75 years vs >75 years), performance status (0–1 vs 2), and prior gastrectomy (yes vs no).Treatment dose and schedulePatients were randomly assigned to receive either capecitabine or S-1 as recommended in the package insert. Capecitabine 2500 mg m−2 was administered orally in two divided doses daily on days 1–14 of a 21-day cycle. The dosage of capecitabine was adjusted or interrupted for treatment-related adverse events of grade 2 or worse based on a previously defined algorithm (Blum et al, 1999). S-1 was given orally two times daily for 28 days, followed by 14 days’ rest. Three dosage levels of S-1 were defined according to body surface area (BSA) as follows: BSA less than 1.25 m2, 40 mg two times daily; BSA, 1.25 to 1.5 m2, 50 mg two times daily; and BSA more than 1.5 m2, 60 mg two times daily. S-1 was temporarily discontinued and the same dose was retried if patients experienced grade 2 nonhaematologic toxicities, grade 3 thrombocytopenia, or uncomplicated grade 4 neutropenia. If the toxicity recurred or grade 3 nonhaematologic toxicities, grade 4 thrombocytopenia, or febrile neutropenia occurred, S-1 was interrupted until the toxicity subsided to grade 1 or less. The BSA-adjusted S-1 dose was then reduced from 120 to 100 mg day−1, from 100 to 80 mg day−1, or from 80 to 50 mg day−1. The subsequent chemotherapy cycle was started only if the ANC recovered to ⩾1500 μl−1 and the platelet count reached ⩾100 000 μl−1, and nonhaematologic toxicity recovered to grade 1 or less. A treatment delay of up to 3 weeks was permitted without dose reduction. If the ANC was ⩾1000 μl−1 but <1500 μl−1 and the platelet count was ⩾75 000 μl−1 but <100 000 μl−1 on the scheduled day 1 of the next cycle after a 1-week delay, treatment could be started with a 25% reduced dose of capecitabine or at the next lowest dose level for S-1. Each treatment was continued until the occurrence of disease progression, unacceptable toxicities, or patient's refusal to continue.Pretreatment and on-treatment evaluationNo more than 2 weeks before study entry, patients underwent the following evaluations: medical history; complete physical examination including ECOG performance status; complete blood count, serum chemistry with electrolyte and coagulation battery; urinalysis; chest X-ray; electrocardiogram; and computed tomography (CT) of the abdomen and pelvis (CT of chest or neck was also performed if indicated). Charlson comorbidity index (CCI) was prospectively calculated before treatment (Charlson et al, 1987). Other investigations, for example, bone scan or bone X-ray, were performed if clinically indicated to document metastatic disease.All patients were reviewed before the commencement of each cycle of chemotherapy. Complete and differential blood counts and serum chemistry were performed before each 21-day cycle for patients receiving capecitabine and before each 42-day cycle for patients receiving S-1. More frequent reviews and monitoring were undertaken if clinically indicated.Compliance with study medications was monitored by questioning patients and counting their remaining pills at each visit. The ratio of the actual dose taken to the prescribed dose was calculated and used to calculate relative dose intensity.All patients completed a quality-of-life (QOL) questionnaire (EORTC QLQ-C30, QLQ-STO22 Korean version) within 14 days before registration, with each even-numbered chemotherapy cycle for patients receiving capecitabine or each chemotherapy cycle for patients receiving S-1, at the end of the study treatment, and every 2 months thereafter. Results on QOL are to be reported in a separate publication.Response and toxicity criteriaTumour response was assessed every two cycles in the capecitabine arm and every cycle for S-1 according to the RECIST criteria. At each of these assessments, the same imaging technique was used as was employed at baseline. National Cancer Institute Common Toxicity Criteria, version 2.0, were used to assess toxicity. Radiographs of all eligible patients were also reviewed extramurally to confirm investigator-designated responses.Statistical analysisThe primary end point was ORR as assessed by external independent review, which was analysed on both an intention-to-treat (ITT, all eligible patients who were randomly assigned) and per-protocol (PP, treated patients eligible and assessable for response without major protocol violations) basis. Patients were considered assessable for response if they had received at least two cycles of chemotherapy in the capecitabine arm and one cycle in the S-1 arm and had had at least one follow-up tumour assessment. However, patients were also considered assessable if they had received less than the predefined number of cycle(s) of chemotherapy due to rapid tumour progression.Simon's optimal two-stage design was used for both treatment arms to allow early termination of inactive arm(s). To test the null hypothesis P0⩽0.1 vs the alternative hypothesis P1⩾0.25, the first stage required at least three or more patients out of 18 to have a confirmed response with α=0.05 and β=0.2 before proceeding to the second stage. In the second stage, 25 assessable patients could be added and if a total of eight or more patients achieved a confirmed response, then the primary end point would have been met.The secondary end points were time-to-progression (TTP), time-to-treatment failure (TTF, including progression, death, or withdrawal), and OS. Kaplan–Meier estimates and the Cox proportional hazard model were used in the analysis of time-event variables. Comparison of the distribution of discrete variables in the two treatment arms were performed by the χ2 test or Fisher's exact test when appropriate, and further evaluated by logistic regression analysis. For continuous variables, the Mann–Whitney U-test for nonparametric data was used. All tests were two sided and a P-value<0.05 was considered as statistically significant. SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.ResultsPatient characteristicsFrom October 2004 to April 2006, 96 patients from nine centres in Korea were enrolled; 49 were randomly assigned to receive capecitabine and 47 to S-1. As shown in Figure 1, five patients were ineligible and were excluded from the analysis. Therefore, the ITT population contained 91 patients. Two patients in the capecitabine arm withdrew consent without study treatment and were excluded from the PP analysis. The baseline characteristics were well balanced between the two treatment arms (Table 1). The median age of patients was 71 years (range, 65–82 years) and the CCI was 1 or more in 37% of patients.Exposure to study medicationIn total, 240 cycles of capecitabine and 144 cycles of S-1 were administered, with a median of five cycles (range, 1–22) in the capecitabine arm and two cycles (range, 1–14) in the S-1 arm. Thirty-four patients in the S-1 arm received 60 mg two times daily and the remaining 11 patients received 50 mg two times daily. The median relative dose intensity per patient was 87.6% (range, 32.3–102.4%) for capecitabine and 96.3% (range, 16.1–102.5%) for S-1 (P=0.003). Although the relative dose intensity for S-1 remained stable, there was a steady decrease in relative dose intensity for capecitabine during treatment (Figure 2). There were more dose reductions (27.5% of cycles and 36.4% of patients) and dose delays (12.7% of cycles and 31.8% of patients) with capecitabine than with S-1 (2.1% of cycles and 11.4% of patients for dose reductions, and 7.6% of cycles and 13.3% of patients for dose delays). Hand–foot syndrome (16.9% of cycles) and neutropenia (4.7% of cycles) were the most frequent reasons for dose reductions in the capecitabine arm. These toxicities did not result in dose reduction in the S-1 arm.The main reason for discontinuing study treatment was disease progression (64% of patients in the capecitabine arm and 71% of patients in the S-1 arm). Other reasons included patient refusal (22.7% for capecitabine and 13.3% for S-1), death (4.5 and 4.4%, respectively), and other concomitant illnesses not associated with study treatment (6.8 and 6.6%, respectively). Only one patient in each treatment group was taken off study treatment because of intolerable treatment-related toxicities: recurrent grade 2 vomiting in the capecitabine arm, and grade 3 abdominal pain in the S-1 arm.Treatment responseThe ORR for both the ITT and PP populations are shown in Table 2. Both treatments met the primary end point of this study. The confirmed ORR for the ITT population was 26.1% for capecitabine (95% CI, 13.4–38.8) and 28.9% for S-1 (95% CI, 15.6–42.1). ORR for the PP population was 27.2% for capecitabine (95% CI, 14.1–40.4) and 28.9% for S-1 (95% CI, 15.6–42.1). The median duration of response in the ITT population was 6.3 months for capecitabine (95% CI, 5.46–7.14) and 8.5 months for S-1 (95% CI, 5.70–11.26), with a hazard ratio (capecitabine/S-1) of 1.3 (95% CI, 0.54–3.20). The reason that patients were not assessed for efficacy are shown in Figure 1. Logistic regression analysis revealed that none of the potential prognostic factors were statistically significant predictive factors for overall response (Table 3).Secondary end pointsAt the last data cutoff date, 15 May 2007, the median follow-up duration was 21.9 months in the capecitabine arm (95% CI, 19.4–24.5) and 21.7 months in the S-1 arm (95% CI, 18.4–25.0). At this time, 73 of the 91 patients had died. Disease progression was the major cause of death and accounted for 95.8% of patient deaths in both treatment groups. Two patients in the capecitabine arm died from non-cancer-related causes (intracranial haemorrhage, n=1; sudden cardiac death, n=1) both of which had no direct causal relationship with study medication. The cause of death in one patient allocated to S-1 was unclear.The median TTP was 4.7 months for capecitabine (95% CI, 3.1–6.4) and 4.2 months for S-1 (95% CI, 1.5–6.9), with a hazard ratio (capecitabine/S-1) of 1.0 (95% CI, 0.6–1.6) (Figure 3). The probability of remaining progression-free at 6 months was 35.8 and 42.2% for the capecitabine and S-1 arms, respectively. The median TTF was 4.3 months for capecitabine (95% CI, 2.8–5.7) and 3.0 months for S-1 (95% CI, 1.5–4.5). The median OS was 9.5 months for capecitabine (95% CI, 7.8–11.3) and 8.1 months for S-1 (95% CI, 4.9–11.4); the hazard ratio (capecitabine/S-1) was 0.9 (95% CI, 0.5–1.4) (Figure 4). The 1-year survival rate was 30.2% for capecitabine and 27.3% for S-1. Cox proportional hazard analyses found that none of the potential prognostic factors were statistically significant predictive factors for OS (Table 3). However, patients who received prior gastrectomy had tendency towards a longer OS according to both univariate and multivariate analyses.SafetyHaematologic and nonhaematologic adverse events are shown in Table 4. The incidence of severe adverse events was very low in both treatment groups. With the exception of hand–foot syndrome and stomatitis, which were more frequently found in the capecitabine arm, the incidence of grade 2 or worse toxicities was similar in the two treatment arms.DiscussionThis is the first randomised trial of two new oral fluoropyrimidines, capecitabine or S-1, in elderly patients with AGC. Although this was a phase II study and did not aim to compare the treatment groups statistically, we could estimate the relative efficacy and safety of the two drugs. Elderly patients may be the best target population for this purpose because single-agent chemotherapy can be a good and safe first-line treatment option for this patient group and it avoids the compounding effects of other agents if combination therapy is used. For the assessment of the primary study end point, ORR, all images were extramurally reviewed and all responses were confirmed. Both agents met the primary end point and seemed to have similar activity in elderly patients with AGC. In the PP population, ORRs of 28.9% with S-1 and 27.2% with capecitabine were observed. The ORR for capecitabine is largely consistent with a previous study performed in patients with AGC (Hong et al, 2004), whereas that for S-1 is somewhat lower than that observed in Japanese trials (Sakata et al, 1998; Koizumi et al, 2000), but is better than that observed in a non-Japanese Asian study (Jeung et al, 2007). Median TTF, TTP and OS were also very similar for both regimens.Capecitabine and S-1 were well tolerated and no treatment-related deaths were reported in this study. Both agents were minimally myelosuppressive and the most frequent haematological toxicity was anaemia. Although one episode of febrile neutropenia occurred in one patient treated with capecitabine, there were no meaningful differences in haematologic toxicities between the two treatment arms. The most frequently observed grade 3/4 nonhaematological toxicities were anorexia and asthenia. Although these toxicities developed in less than 10% of patients, they had a tendency to become more clinically significant as treatment progressed. The only notable differences in grade 2 or worse nonhaematologic adverse events between S-1 and capecitabine were the greater incidences of hand–foot syndrome and stomatitis among patients treated with capecitabine. There was a steady decrease in the relative dose intensity of capecitabine during treatment caused by treatment delays or dose reductions mostly because of hand–foot syndrome, whereas that of S-1 remained steady (Figure 2). Considering these findings, we suggest that a dose of 1000 mg m−2 two times daily, lower than that recommended, would be appropriate for capecitabine in elderly patients (Bajetta et al, 2005), whereas the recommended dose of 40 mg m−2 two times daily would be acceptable for S-1. These findings suggest that dose escalation of S-1 may be possible in younger patients.The rates of some key adverse events reported with capecitabine in our study, as well as in other phase II studies performed in Japan or Korea (Hong et al, 2004; Lee et al, 2004; Hyodo et al, 2006), appear to differ from those reported in phase III multinational studies (Cassidy et al, 2002; Twelves et al, 2005). The rates of grade 3 hand–foot syndrome (6–13% in Asian phase II studies vs 17% in multinational phase III studies) and grade 3/4 diarrhoea (2–5 vs 11–13%) tend to be lower, and the rate of grade 3/4 neutropenia (0–8 vs 1–2%) similar or higher. These observations are consistent with the recent work of Haller et al (2008) who reported regional differences in the tolerability of fluoropyrimidines. They found that the risk of adverse events was lowest in patients from East Asian centres, except for grade 3/4 neutropenia, which was most likely to occur in patients from East Asia. No safety data regarding S-1 monotherapy from the United States or Europe are yet available.According to recent SEER data from the United States, 65.5% of gastric cancers are diagnosed in patients older than 65 years: the median age at diagnosis of gastric cancer was 71 years and the median age of gastric cancer-related death was 74 years (Ries et al, 2007). However, because elderly patients are generally excluded from cancer chemotherapy clinical trials, data to guide the treatment of older patients with AGC in an evidence-based fashion are lacking (Lewis et al, 2003; Murthy et al, 2004; Trumper et al, 2006; Lichtman et al, 2007b). The recently reported SPIRITS trial and a meta-analysis on first-line chemotherapy in patients with AGC demonstrated a statistically significant benefit in OS for 5-FU-based combination chemotherapy compared with single-agent chemotherapy (Wagner et al, 2006; Koizumi et al, 2008). Trumper et al (2006) suggested that elderly (⩾70 years) patients without significant co-morbidities should be treated with the same regimens as younger patients with AGC based on a retrospective review of three UK multicentre randomised trials. However, the SPIRITS trial only involved patients of less than 75 years, and extrapolation of the results from retrospective reviews or meta-analyses to the elderly patients must be done with caution because of the following limitations: (1) a small, approximately 1-month, survival advantage observed in the meta-analysis was achieved at the expense of increased toxicity (Wagner et al, 2006); (2) chemotherapy-related toxicities, such as neutropenia, anaemia, stomatitis and diarrhoea, occurred more frequently in the elderly (Trumper et al, 2006); (3) the early drop out rate was significantly higher and 5-FU dose intensity was significantly lower in the elderly when treated with combination chemotherapy containing 5-FU and cisplatin (Trumper et al, 2006); and (4) QOL, which could be impaired as the intensity of chemotherapy increases, has not been studied sufficiently. Ideally, standard treatment of AGC in elderly patients should not be based solely on retrospective subset analyses of prospective trials, and elderly specific trials are needed to define the optimal treatment for these patients (Perrone et al, 2002; Jatoi et al, 2005). Considering the ORR, OS and safety results, our study provides evidence that elderly patients with AGC could benefit from capecitabine or S-1 monotherapy with minimal adverse events.In conclusion, our study indicates that both capecitabine and S-1 are safe, well tolerated and efficacious in older patients with AGC. Oral agents, such as capecitabine or S-1, may have particular appeal in the management of cancer in this patient population. An additional trial is needed to clarify the potential predictive factors for drug selection and to establish the effectiveness of various combinations, including molecular targeted agents, in older patients with AGC.\n\nREFERENCES:\n1. 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+{
+    "id": "PMC2528007",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528007\nAUTHORS: A. Louise McCormick, Linda Roback, Edward S. Mocarski\n\nABSTRACT:\nViruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle.\n\nBODY:\nIntroductionCell death is central to viral infection, as an evolutionarily-conserved means to eliminate intracellular pathogens and as a way that lytic viruses mediate release of progeny. Human cytomegalovirus (CMV), the major infectious cause of birth defects as well as an important cause of opportunistic disease worldwide [1], remains cell-associated during productive replication. Release of progeny virus depends upon the exocytic pathway [1] and continues until cells die via a poorly understood fragmentation process. CMV is well-armed to modulate cell-intrinsic as well as extrinsic innate and adaptive host clearance pathways [1]. The product of the UL37x1 gene, vMIA, a potent suppressor of apoptosis [2]–[4], also controls the timing of infected cell death [5]–[7]. Premature death in vMIA-mutant virus-infected cells reduces the period of progeny release by three to four days [5]–[7] without affecting cell-to-cell spread [5]. All vMIA-mutant viruses exhibit this premature death phenotype, but the involvement of caspases and the impact on viral yield varies with CMV strain. AD169varATCC strain (AD-BAC) depends upon vMIA to a greater extent [6],[7] than TownevarATCC (Towne-BAC), although vMIA prolongs the period of viral replication and release in both strains [5]. Importantly, vMIA from either strain retains the capacity to block caspase-dependent apoptosis [5]. The caspase-independent death pathway that is blocked by vMIA is not known. Other cell death suppressors are encoded by CMV [1], but, aside from vMIA, only UL38 has been implicated in control of infected fibroblast death to prolong replication [8],[9]. Studies to date reveal a complexity of infected cell death and a need for a more complete understanding of events that naturally terminate CMV infection.Major pathways associated with death (apoptosis, necrosis, and autophagy) are triggered by specific host cell and immune system initiators and exhibit characteristic molecular events and cell morphological changes [10]–[12]. Proteases in the caspase, calpain, lysosomal cathepsin, and proteasomal serine protease classes are central to the execution of various death pathways. The fact that the premature death induced by vMIA-mutant CMV is resistant to inhibitors of caspases, cathepsins, and calpains [5] suggests a novel programmed pathway distinct from characterized death pathways [11],[13]. For those viral strains that have been characterized, infected cell death initiates approximately 7 to 10 days after infection of fibroblasts. In contrast, the premature death that occurs in vMIA mutant virus infection initiates 3 to 4 days postinfection [5]–[7]. The 12–24 h timing of individual cell fragmentation, association with cytopathic effect (CPE), and nominal impact of vMIA [5] all suggest that the final stages of productive replication terminate with a CMV infected cell-specific programmed cell death (cmvPCD).Many DNA viruses encode antiapoptotic functions that sustain replication in the face of cell-intrinsic defenses [14]–[16]. vMIA equips CMV to counteract intrinsic host clearance pathways leading to cell death [5]–[7]. As an outer mitochondrial membrane protein, vMIA sits in a central position analogous to antiapoptotic Bcl-2 family members Bcl-2 and Bcl-xL\n[2], and prevents the formation of a mitochondrial permeability transition pore, release of cytochrome c and proapoptotic factors into the cytoplasm, and activation of executioner caspases. Unlike antiapoptotic Bcl-2 family members [2],[5],[17], vMIA lacks Bcl-2 homology domains but depends on an antiapoptotic domain that mediates interaction with GADD45 family members [18],[19]. vMIA also recruits BAX to mitochondria [20],[21] and disrupts mitochondrial networks [22]. This disruption normally accompanies BAX oligomerization at the outer mitochondrial membrane [23],[24], although vMIA mutants that fail to bind BAX continue to disrupt networks [25]. The vMIA-dependent recruitment of BAX does not lead to the formation of a transition pore complex or release of proapoptotic mediators [20],[21]. The contribution of BAX oligomerization or mitochondrial network disruption to cell death suppression remains to be investigated. Although both of these events are signs of apoptosis [23],[24],[26],[27] neither mitochondrial network disruption [28] nor BAX oligomerization [29],[30] are sufficient to induce apoptosis. These alterations are also associated with vMIA-mediated suppression of cell death during viral infection where the pathway(s) of death are not fully understood.Consequences of mitochondrial release of proapoptotic mediators have been extensively studied [10], [31]–[33]. Cytochrome c controls apoptosome formation and downstream executioner caspase activation. Endonuclease G and apoptosis-inducing factor (AIF) promote nuclear events. Mitochondrial release of Smac/DIABLO and HtrA2/Omi overcomes the activity of inhibitor of apoptosis proteins (IAPs). The HtrA2/Omi proenzyme is processed within the mitochondria, removing a mitochondrial targeting sequence (amino terminal 33 amino acids) and a transmembrane domain [34]–[36]. Mature, active HtrA2/Omi resides in the intermembrane space, and is released into the cytoplasm through the transition pore complex at the same time as cytochrome c. Release of the serine protease HtrA2/Omi from mitochondria can result in two downstream effects: (1) cleavage of IAPs and an ultimate increase in caspase-dependent death and (2) trigger IAP-independent, caspase-independent death [37]. This latter pathway is also induced by extramitochondrial overexpression of HtrA2/Omi in the cytoplasm [35], [37]–[40]. The role of this serine protease as an inducer of cell death [38],[39] in mammalians seems opposite the role of the founding member of this protein family as a pro-survival serine protease in eubacteria [41]–[43]. Here, we demonstrate the central role of HtrA2/Omi executing a serine protease-dependent pathway that is controlled by vMIA during infection.ResultsPremature cell death in the absence of vMIAWe evaluated cmvPCD during wild type (wt) virus (Towne-BAC, a GFP-expressing virus [44]) infection by scoring morphological changes in cells during replication (Fig. 1, supplemental Fig. S1). Termination of infection was associated with the accumulation of GFP-positive cell debris that remained associated with the monolayer (Fig. 1A). Cell fragmentation and death was first observed at 120 h postinfection in a small percentage of GFP+ foci (Fig. 1A, Fig. S1). GFP+ dead cell debris was observed only in foci (Fig. S1). Almost all (>90%) foci showed evidence of fragmentation by 240 h postinfection (Fig. 1C). Thus, cmvPCD occurred very late in infection, after maturation and release of progeny virus had peaked. GFP+ debris was observed much earlier during infection with vMIA null mutant virus, ΔUL37x1 (Fig. 1C), although the fragmentation process appeared similar to wt virus (Fig. 1B, Fig. S1). As previously reported [5], a majority (70%) of mutant virus foci contained debris by 120 h due to the single GFP+ cells that started to fragment between 72 and 96 h postinfection prior to the formation of foci (Fig. S1). There was a gradual increase in foci containing fragmented cells between 120 and 192 h postinfection such that, by 192 h, >90% of foci contained fragmented cells (Fig. 1C). This was consistent with our previous report showing both viruses spread with equivalent efficiency but that ΔUL37x1 induced premature caspase-independent death [5].10.1371/journal.ppat.1000063.g001Figure 1Pattern and timing of cmvPCD in Towne-BAC and ΔUL37x1-infected cells.Images of infected cell foci (multiplicity of infection [MOI] of 0.01) showing cmvPCD in Towne-BAC (A) and ΔUL37x1 (B) infected cells at 120 h postinfection (hpi). GFP expression by the viruses was used to identify foci of infected cells. Original magnification ×400. (C). The mean percentages (±standard deviation) of GFP+ foci with fragmented cells based on counting 400 infected foci each day. Infection was with a MOI of 0.0001. The mean±sd is depicted in all figures, except where indicated.ΔUL37x1-initiated death is a late phase eventIntact GFP+ ΔUL37x1-infected cells appeared to fragment before virus spread to form foci (Fig. 1C, Fig. S1) [5]. To determine whether intact ΔUL37x1-infected cells released progeny virus before fragmenting, we evaluated the formation of immediate early nuclear antigen positive (IE+) foci by immunofluorescence. Infected cells became IE+ earlier than they became GFP+ (Fig. 2). IE+ foci surrounding single GFP+ cells were detected at 72 h postinfection (Fig. 2A–E), when a majority (>50%) of GFP+ cells were still intact (Fig. 2A, 2D). Whether intact or fragmented, >99% of ΔUL37x1-infected cells produced foci by 120 h postinfection [5]. Thus, most virus spread occurs before cells fragmented (Fig. 2A, 2D), suggesting that, like wt, mutant virus is released before infected cells die (Fig. 1, Fig. S1).10.1371/journal.ppat.1000063.g002Figure 2cmvPCD follows maturation and release in Towne-BAC and ΔUL37x1-infected cells.Representative fluorescent images of immediate early positive (IE+) nuclei in foci surrounding an intact, live (A) or fragmented, dead GFP+ cell (B). (GFP = green, indirect fluorescence of IE antigen = red; original magnification ×200). (C–E) Percentage of total GFP+ (C), live GFP+ (D), or dead (debris) (E) GFP+ cells forming IE1+ foci at 72 and 96 h postinfection with ΔUL37x1 or Towne-BAC (MOI of 0.001). A total of 300 GFP+ cells per virus were evaluated at each time for the experiment depicted. (F) Percentage of IE+ cells (live) at 96 h postinfection with ΔUL37x1 (MOI of 0.003), comparing untreated cultures to cultures treated with 300 µg/ml PFA or 20 µM BDCRB from 1 h postinfection. Data are from a representative experiment in which a total of >1000 infected cells/foci were evaluated for each condition.To confirm that the death of mutant virus-infected cells was dependent on late phase events, the DNA synthesis inhibitor phosphonoformate (PFA) or the DNA encapsidation inhibitor 2-bromo-5,6-dichlorobenzimidazole (BDCRB) was added at the time of CMV infection and cell fate was scored by staining for IE+ cells at 96 h postinfection (Fig. 2B, 2F). PFA inhibits late gene expression, including GFP, while BDCRB blocks virion maturation but allows late gene expression to proceed [45],[46]. Untreated control or BCDRB-treated infected cultures appeared similar, whereas PFA-treated cultures contained single IE+ cells that remained GFP-. Only about 30% of GFP+ cells or debris in untreated ΔUL37x1-infected cells were IE+ at 96 h postinfection (Fig. 2F). The remaining 70% were no longer IE+, suggesting that IE expression was lost as cells fragmented. BDCRB-treated cells exhibited a pattern similar to untreated cells, suggesting that fragmentation was triggered by events prior to DNA encapsidation. All infected cells in PFA-treated cultures remained IE+ (Fig. 2F) and did not fragment, suggesting that initiation of cell death was dependent on events that followed viral DNA synthesis. Thus, initiation of death was dependent on cellular changes associated with viral DNA replication and/or late phase gene expression.cmvPCD proceeds rapidly through fragmentation to deathWe evaluated cell morphology [11] associated with death in ΔUL37x1 and wt virus-infected cells (Fig. 3). Late in CMV infection, inclusions form within enlarged cells coincident with replication and maturation processes that take place in the nucleus as well as in the cytoplasm. At 72 h postinfection, ΔUL37x1 and wt virus-infected cells exhibited similar nuclear and cytoplasmic inclusions [1],[47],[48] as well as enlarged cell CPE (Fig. 3A, 3F and Fig. S2) [5]. Stain for total nuclear DNA revealed a diffuse pattern (Fig. 3F, Fig. S2) that became distorted (Fig. 3G) and progressed through shrinkage and collapse and loss of nuclei (Fig. 3H–J) as cells fragmented (Fig. 3C–E). A similar process accompanied fragmentation in mutant or wt infected cells. The fragmentation process produced cell debris (Fig. 3E) lacking signs of DNA (Fig. 3J). Loss of nuclei scored by DNA stain or IE antigen (Fig. 2) was similar. Cell debris remained GFP+ and unstained by ethidium homodimer (Fig. S3), suggesting a non-necrotic death. Fragmentation was not synchronous in infected cultures, such that only 10% of infected cells exhibited intermediate fragmentation patterns (e.g. Fig. 3B–D) at any time (Fig. 3K and Fig. S1). The same types of morphological changes that started at 72 h in ΔUL37x1-infected cultures started at 120 h postinfection in Towne-BAC-infected cells (Fig. S1 and S4). The fragmentation of GFP+ cells (Fig. 1), loss of IE+ cells (Fig. 2) and loss of DNA+ nuclei (Fig. 3) were all characteristic of cmvPCD in wt and premature death in mutant virus-infected cells.10.1371/journal.ppat.1000063.g003Figure 3cmvPCD proceeds through cell fragmentation and nuclear collapse in the absence of chromatin condensation.Representative images showing stages of infected cell fragmentation and death in ΔUL37x1 infected cultures (collected at 72 h postinfection). Fluorescent micrographs of GFP+ virus-infected cells (A–E) and Hoechst-stained nuclei (F–J) associated with fragmentation. (A, F) Cytomegalic infected cell without signs of fragmentation, (B, G) initial stages of fragmentation with intact nucleus, (C, H) fragmented cell body with collapsed nuclear body, (D, I) fragmented cell body with residual nuclear body, and (E, J) fragmented cell debris without residual nuclear material. Original magnification ×1000. (K) Counts of GFP+ intact infected cells (Intact Cell), Hoechst positive fragmented cell bodies (Fragmented Cell), and Hoechst negative fragmented cell debris (Debris) at 72 and 96 h postinfection with ΔUL37x1 (MOI of 0.002). Data are from a representative experiment in which a total of 750 infected cells/foci were evaluated for each time.Serine proteases induce cmvPCDPrevious work showing that caspases, cathepsins, or calpains were not involved in ΔUL37x1-initiated premature death [5], lead us to evaluate the contribution of cellular serine proteases to this process. We started by assessing the impact of a broad-spectrum inhibitor, TLCK [49]–[62], because this inhibitor does not affect the viral maturational serine protease at concentrations that are sufficient to block cellular serine proteases [63]. Addition of TLCK (11, 33, or 100 µM) to infected cultures at 30 h lead to a concentration-dependent reduction in cell fragmentation at 72 h postinfection (Fig. 4A). These concentrations of inhibitor did not reduce virus yields (Fig. 4B). Thus, TLCK inhibited premature cell death without any impact on virus replication. When TLCK was added at 30 h and fragmented cells were counted at 72, 96, and 120 h postinfection (Fig. 4C), cell death was reduced approximately twofold, suggesting that serine proteases play a central role in the timing of fragmentation. Despite experiment-to-experiment variability in the levels and rate of fragmentation death observed between 72 and 120 h postinfection, TLCK consistently inhibited this process (Fig. 4A and C) and increased the proportion of live, intact cells while absolute numbers of GFP+ cells or debris remained the same (Fig. 4D). This result implicated serine proteases early in the premature death induced by mutant virus.10.1371/journal.ppat.1000063.g004Figure 4The serine protease inhibitor TLCK prevents ΔUL37x1-induced premature cmvPCD.(A) Percentage of dead GFP+ infected cells at 72 h postinfection in untreated cultures (U) or cultures treated with 11, 33, or 100 µM TLCK from 30 h postinfection with ΔUL37x1 (MOI of 0.001). A total of 300 cells/foci were evaluated at each condition. (B) Virus yield at 120 h postinfection from cell cultures left untreated (U) or treated with 11, 33, or 100 µM TLCK from 30 h postinfection with ΔUL37x1 or Towne-BAC (MOI of 0.001). (C) Percentage of fragmented GFP+ cells at 72, 96, or 120 h postinfection with ΔUL37x1 (MOI of 0.005) in control cultures or cultures treated with 33 µM TLCK from 30 h postinfection. A total of 1450 cells/foci were evaluated at each condition. (D) Absolute numbers of live or dead GFP+ cells at 96 h postinfection with ΔUL37x1 (MOI of 0.001) in untreated cultures or cultures treated with 33 µM TLCK from 30 h postinfection. (E) Percentage of dead GFP+ cells at 96 h postinfection with ΔUL37x1 (MOI of 0.005) in untreated cultures or cultures treated with 33 µM TLCK, 25 µM zVAD.fmk, or 0.05% DMSO, added from 30 h postinfection. A total of 300 cells/foci were evaluated at each condition. (F) Percentage of live cells at 48 h postinfection with AD169varATCC (MOI of 3) following treatment with anti-Fas antibody to induce apoptosis in control cultures or cultures treated with 6, 12, or 25 µM zVAD.fmk, added at 24 h postinfection.Previously, we reported that the pan-caspase inhibitor zVAD.fmk had no effect on ΔUL37x1-induced premature death [5]. To determine whether caspases influenced death levels when serine proteases were inhibited, we applied zVAD.fmk alone as well as in combination with TLCK. The caspase inhibitor did not influence the serine protease-dependent process (Fig. 4E). In contrast, zVAD.fmk showed the expected [5],[6],[64] inhibition of apoptosis induced in CMV strain AD169varATCC infected cells (Fig. 4F). Thus, these data imply that cmvPCD is controlled by serine proteases that work independent of caspases.The serine protease HtrA2/Omi initiates cmvPCDTo determine the timing of serine protease activity in controlling premature death, TLCK was added to ΔUL37x1-infected cells at 30, 54, or 78 h. Addition of TLCK at each of these times was found to dramatically reduce the level of death at 96 h postinfection (Fig. 5A). These results suggest serine proteases act within 24 h of fragmentation (Fig. 4D) and demonstrated the importance of these proteases late in infection. Taken together with data on timing of the death stimulus (Figs. 2 and 3, and [5]), serine proteases active late in CMV infection may either trigger or play an intermediary role in the cell death pathway. To determine the timing of serine protease activity in wt virus-induced cmvPCD, TLCK was added at 30, 54, and 78 h (Fig. 5B). Addition of TLCK at each of these times effectively reduced cell fragmentation at 144 h postinfection, suggesting that proteases active after the 78 h time period played a critical role during wt virus infection as well (Fig. 5B). These data demonstrate a common serine protease cell death pathway terminates mutant or wt virus infection, and demonstrate that the premature death in mutant virus infected cells follows a similar pathway as cmvPCD. Differences in timing show the importance of vMIA control in the timing of cmvPCD.10.1371/journal.ppat.1000063.g005Figure 5Inhibitors of serine protease HtrA2/Omi suppress cmvPCD.(A) Percentage of dead GFP+ cells at 96 h postinfection with ΔUL37x1 (MOI of 0.01) in cultures treated with 100 µM TLCK from 30, 54, or 78 h postinfection relative to untreated cultures (U). 3000 cells/foci were evaluated at each condition. (B) Percentage dead GFP+ cells at 144 h postinfection with Towne-BAC (MOI of 0.03). 9000 cells/foci were evaluated at each condition. (C) Percentage of dead GFP+ cells at 96 h postinfection with ΔUL37x1 in cultures treated with 10 µM UCF-101 from 30, 54, or 78 h postinfection relative to untreated cultures (U). 3000 cells/foci were evaluated at each condition. (D) Percentage of dead GFP+ cells at 144 h postinfection with Towne-BAC (MOI of 0.03). A total of 9000 cells/foci were evaluated at each condition. Data in A–D is depicted relative to untreated controls, which were assigned a value of 100%. (E) Yields of virus at 168 h postinfection (MOI of 0.01) with ΔUL37x1 or Towne-BAC in untreated cultures or cultures treated with UCF-101 from 30 or 54 h postinfection.One mitochondrial serine protease, HtrA2/Omi, has been implicated in cell death pathways and exhibits sensitivity to TLCK [40], [65]–[67]. The specific HtrA2/Omi inhibitor UCF-101 [68] was added to ΔUL37x1- or Towne-BAC-infected cultures (Fig. 5C–E) at a concentration (10 µM) anticipated to minimize a previously recognized impact on other cellular targets [69]. UCF-101 reduced death when added to ΔUL37x1 or Towne-BAC-infected cultures at 30 or 54 h, implicating HtrA2/Omi as a mediator of cmvPCD (Fig. 5C–D). Although UCF-101 added at 54 h reduced death of ΔUL37x1-infected cells at 96 h postinfection, addition at 78 h was ineffective (Fig. 5C). Towne-BAC-associated death at 144 h was reduced by UCF-101 added as late as 102 h, but not when added at 126 h (Fig. 5D). UCF-101 treatment did not reduce viral yields (Fig. 5E). Most importantly, these data suggest that events over the 24 to 48 h preceding fragmentation of cells are influenced by HtrA2/Omi, regardless of whether considering the premature cmvPCD in mutant virus infected cells or cmvPCD in wt infection. The differences between UCF-101 and TLCK addition at 78 h (Fig. 5A) may be due to the effectiveness of these inhibitors on HtrA2/Omi or to additional serine proteases that contribute to cmvPCD. Overall, these data demonstrate that UCF-101 specifically reduces infected cell death and implicate the serine protease HtrA2/Omi in the pathway. Further, these data implicate HtrA2/Omi as a target of vMIA modulation.HtrA2/Omi levels increase late in infectionTo determine the impact of mutant or wt virus infection on HtrA2/Omi expression levels and subcellular localization as well as to investigate any impact of vMIA on HtrA2 expression, immunoblot analysis was carried out on Towne-BAC infected cells (Fig. 6). Levels of mature 36 kDa HtrA2/Omi levels were similar to uninfected cells at 24 h, but increased by 48 h and continued to accumulate over the course of infection (Fig. 6A–B). Comparisons of mutant and wt infected cells showed similar accumulation of HtrA2 by 48 h (Fig. 6B, Fig. S5). Premature cmvPCD initiated in mutant virus-infected cells prevented comparisons by immunoblot later in infection; however, immunofluorescence analyses at 96 h postinfection confirmed the dramatically increased HtrA2/Omi levels in mutant or wt virus-infected cells (Fig. 6C–H and Fig. S6). HtrA2/Omi colocalized with the mitochondrial membrane potential marker MitoTracker Red (Fig. 6C, 6F and Fig. S6) at late times of infection with either virus. These data indicate that HtrA2/Omi levels increase within mitochondria before the initiation of cmvPCD. vMIA does not alter expression pattern or mitochondrial localization of this protease but nevertheless prevents death.10.1371/journal.ppat.1000063.g006Figure 6HtrA2/Omi expression increases following CMV infection.(A–B) Immunoblot analyses of HtrA2/Omi in lysates of mock-infected HF (M) or HF infected (MOI of 3) with Towne-BAC or ΔUL37x1 for 24, 48, 72, 96, or 120 h and control cell lysates from HeLa cells transfected with HtrA2/Omi expression plasmid (+) or control plasmid (−). The expected migration of the 49 kDa immature, pro-HtrA2/Omi and 36 kDA mature, HtrA2/Omi was calculated based on migration of molecular weight markers (not shown). Immunoblot detection of HtrA2, β-actin and IE1 are shown. (C–N) Immunofluorescent images of HtrA2/Omi (C, F) and cytochrome c (I, L) (red), and GFP fluorescence (green) (D, G, J, M) and merged images (E, H, K, N) at 72 h (C–H) or 96 h (I–N) postinfection (MOI of 0.001) with ΔUL37x1 (C–E, I–K) or Towne-BAC (F–H, L–N). Original magnification ×1000.Mitochondria in wt CMV infected cells followed the expected [22] reticular to punctate transition associated with disruption of mitochondrial networks (Fig. 6F, 6L, and Fig. S6) and mutant virus-infected cells retained a reticular pattern (Fig. 6C, 6I and Fig. S6) when stained for HtrA2/Omi, cytochrome c, mitochondrial HSP (mtHSP70), or MitoTracker Red. MitoTracker Red staining indicated that mitochondria retained a similar membrane potential despite this difference in morphology due to vMIA (Fig. S6). When the kinetics of the reticular to punctate transition was evaluated in Towne-BAC-infected cells, almost all (≥90%) of cells contained reticular mitochondria at 48 h, but transitioned to punctate by 96 h. In contrast, ΔUL37x1-infected cells retained a reticular morphology (≥80%) throughout infection. These data suggest that a vMIA-dependent process disrupts reticular mitochondria beginning at 48 h postinfection and this change in mitochondrial organization may contribute to cell survival. Despite this striking difference in mitochondria, the organelles of the secretory apparatus that form the viral assembly compartment at late times of infection [48],[70] were similar in either virus infection (Fig. S2, Fig. S6). Thus, disruption of mitochondrial networks by wt virus may contribute to control of HtrA2/Omi-dependent death and the failure of mutant virus to induce these changes may lead to premature HtrA2/Omi-dependent death.We sought to determine whether mitochondria released cytochrome c prior to premature cmvPCD. Cells that had not yet started to fragment all showed reticular cytochrome c staining (Fig. 6 and Fig. S6) whereas diffuse staining was detected only as cells became highly fragmented (Fig. S7). These data suggest that release of cytochrome c follows the fragmentation that characterizes cmvPCD.HtrA2/Omi overexpression early after infection blocks CMV replicationTo directly address the impact of HtrA2/Omi overexpression on the cell fate, full-length HtrA2/Omi as well as a catalytic site mutant (HtrA2S306A) [34] were transiently expressed in uninfected and virus-infected cells. Initially, expression levels and impact on uninfected cell viability were assessed (Fig. 7A–H, Fig. S8). HtrA2/Omi (or mutant HtrA2/Omi) overexpression did not induce death in uninfected HFs (Fig. 7H) or HeLa cells (Fig. S8), consistent with published characterization of full-length protease [37]. Immunofluorescence patterns revealed the expected mitochondrial localization at 48 h post transfection (Fig. 7A–F), and immunoblot analyses using HeLa cells indicated equivalent expression levels of the wt and mutant protease (Fig. 7G). To determine the impact of HtrA2/Omi overexpression on infected cells, Towne-BAC or ΔUL37x1 were cotransfected with HtrA2/Omi or HtrA2S306A expression plasmids (Fig. 7I) and assessed for spread to form foci [71]. By 10 days posttransfection, wt and mutant BACmids had produced comparable numbers of plaques, as expected [5]. Cotransfection of HtrA2/Omi expression plasmid reduced the plaquing efficiency >10-fold compared to vector control or HtrA2S306A mutant (Fig. 7I). These data show that overexpression of catalytically active HtrA2/Omi prevents plaque formation independent of vMIA expression.10.1371/journal.ppat.1000063.g007Figure 7Transient overexpression of HtrA2/Omi induces early death in CMV-infected but not uninfected cells.(A–F) Immunofluorescent images of HtrA2/Omi (A–C) and GFP fluorescence (D–F) following cotransfection of HF with GFP expression plasmid together with empty vector, HtrA2/Omi, or HtrA2S306A expression plasmids. Original magnification ×400. (G) Immunoblot depicting 49 kDa, immature and 36 kDA, mature forms of HtrA2/Omi and HtrA2S306A as well as β-actin control in transfected HeLa cell lysates. (H) Percentage of live HFs at 120 h post cotransfection of GFP expression plasmid with vector, HtrA2/Omi, or HtrA2S306A expression plasmids. (I) Number of viral plaques 10 days following cotransfection of ΔUL37x1 or Towne-BAC DNA (500 ng) with 800 ng vector, HtrA2/Omi, or HtrA2S306A expression plasmids. (J) Number of live (intact) cells in cultures at 48 or 72 h following transfection with Towne-BAC DNA (300 ng) alone or with 333 ng of HtrA2 or HtrA2S306A expression plasmids. (K) Number of live (intact) cells (±range) at 48 or 72 h post transfection with Towne-BAC DNA (500 ng) and 300 ng of vector or HtrA2/Omi expression plasmid with or without addition of 10 µM UCF-101 from 6 h.To determine whether the reduction in plaguing efficiency following overexpression of HtrA2/Omi was due to cell death induction, the fate of individual cells was monitored (Fig. S9). When Towne-BAC was cotransfected with HtrA2/Omi or HtrA2S306A, individual GFP+ cells were observed at 48 h, although even at this time the levels could be lower in cells receiving the protease active form (Fig. 7J–K). HtrA2/Omi/GFP+ cells began to fragment by 72 h posttransfection (Fig. 7J) and were lost from cultures by 168 h (Fig. S9). HtrA2/Omi overexpression-induced death required the active protease, based on the failure of HtrA2S306A to induce death (Fig. 7J) as well as on the ability of the inhibitor UCF-101 to block HtrA2/Omi overexpression-induced death (Fig. 7K). The numbers of GFP+ cells (Fig. 7J) or plaques (Fig. 7I) that formed following cotransfection of Towne-BAC with HtrA2S306A could not be distinguished from transfection of Towne-BAC with vector. These data show that overexpression of catalytically active HtrA2/Omi induces infected cell death that is independent of vMIA expression. The sensitivity of virus-infected cells and lack of impact on uninfected HFs (Fig. 7H) supports the specific role of HtrA2/Omi in a novel cell death pathway in CMV-infected cells.A role of vMIA in HtrA2-induced death was investigated using the cotransfection assay carried out using lower doses of expression plasmids as well as using vMIA-expressing cells. Cotransfection of HtrA2/Omi expression plasmid at a 25 or 30-fold lower level revealed a differential impact on these viruses (Fig. 8A), where Towne-BAC exhibited a greater resistance to HtrA2/Omi-induced death. These conditions were also employed to demonstrate that vMIA overexpression overcame HtrA2/Omi-induced death (Fig. 8A). To address the role of vMIA further, HFs as well as HFs stably transduced with retroviruses expressing Myc-tagged vMIA or mutant protein [18] vMIAmut (Fig. 8B–C) were infected. As expected [5], vMIA-HFs suppressed the premature cmvPCD when assessed at 96 and 120 h postinfection, whereas vMIAmut-HF or nontransduced control HFs did not (Fig. 8B). These data suggest the intact antiapoptotic domain of vMIA is required to control premature cmvPCD. The experimental plating efficiency of ΔUL37x1 virus was the same on either cell line (Fig. S10 and [5]). These results were consistent with a role for vMIA in controlling kinetics of cmvPCD and suggest that similar functional domains of vMIA are required in suppression of apoptosis or HtrA2-dependent cmvPCD. Immunoblot analyses were used to compare transduced vMIA (or vMIAmut) levels relative to native viral expression (Fig. 8C). The lower levels of transduced gene expression likely contribute to the death suppression observed (Fig. 8B). Rescue viruses derived from ΔUL37x1 confirmed that an intact UL37x1 locus is sufficient to completely control premature death, mitochondrial organization, and viral yield (Fig. 8D, Fig. S10). Overall, these data confirm the critical role of vMIA as a determinant of cmvPCD when induced by overexpression of HtrA2/Omi transfection or during the late phase of infection. Thus, ΔUL37x1 infection sensitizes to the prodeath impact of HtrA2/Omi, and vMIA controls HtrA2/Omi prodeath pathways during wt CMV infection.10.1371/journal.ppat.1000063.g008Figure 8vMIA suppression of HtrA2/Omi-dependent cmvPCD.(A) Average number of live cells (±range) in cultures at 72 h post transfection of Towne-BAC DNA (500 ng) with 12 ng of HtrA2/Omi expression plasmid or control vector or post transfection of ΔUL37x1 DNA (500 ng) with 12 ng of HtrA2/Omi expression plasmid in the absence or presence of 15 ng vMIA-encoding plasmid or 12 ng of control vector in the absence or presence of 15 ng vMIA-encoding plasmid. (B) Percentage of dead cells at 96 and 120 h postinfection (MOI of 0.0001) with ΔUL37x1 in HF, vMIAmut-HF, or vMIA-HF cultures. 400 cells/foci were evaluated for each condition. (C) Immunoblot detection of native vMIA (vMIA-nat.), vMIA-myc, and vMIAmut-myc in cell lysates collected at various h postinfection (MOI of 3) with ΔUL37x1 or Towne-BAC. Top, α-myc antibody; bottom, α-vMIA antibody. (D) Percentage of dead cells at 72, 96, 120, and 148 h postinfection (MOI of 0.0001) with ΔUL37x1, Towne-BAC, ΔUL37x1R1, or ΔUL37x1R2. 100 cells/foci were evaluated for each condition.vMIA antiapoptotic function remains intact and independent of HtrA2/Omi during infectionIn order to determine whether the antiapoptotic activity of vMIA is preserved in cells where HtrA2/Omi is overexpressed, we performed experiments with HtrA2/Omi expression constructs in HeLa cells exposed to Fas-mediated apoptosis (Fig. 9) [2],[19]. Immunofluorescence analyses showed expected levels and localization of HtrA2/Omi and vMIA in transfected cells (Fig. 9A–I). These analyses indicated that vMIA and HtrA2/Omi (or HtrA2S306A) colocalize with mitochondria under all conditions. Introduction of HtrA2/Omi or mutant expression constructs did not influence the antiapoptotic activity of vMIA (Fig. 9J–L), consistent with previous work showing vMIA-dependent antiapoptotic function is active at late times of infection [5],[6]. Together, these data suggest that HtrA2/Omi does not interfere with vMIA-mediated control of apoptosis.10.1371/journal.ppat.1000063.g009Figure 9HtrA2/Omi overexpression does not impede vMIA antiapoptotic activity.(A–I) Immunofluorescent images of vMIA (A–C) or HtrA2/Omi (D–F) as well as Hoechst staining (G–I) in HeLa cells following transfection with vMIA together with vector, HtrA2/Omi, or HtrA2S306A expression plasmids. Original magnification ×400. (J–L) GFP+ cell number as a percentage of control wells (vector) of HeLa cells cotransfected with vMIA (1 µg) or GFP expression plasmids and 1 µg (J), 2 µg (K), or 3 µg (L) quantities of vector, HtrA2/Omi, or HtrA2S306A DNA. GFP+ cell numbers in vMIA plus vector wells were assigned values of 100%.Increased serine protease accumulation in cells undergoing premature cmvPCDTo directly visualize levels of serine proteases in infected cells, the fluorescent reagent sulforhodamine 101-leucine chloromethyl ketone (SLCK) was used to reveal the distribution and activity of serine proteases [72] in ΔUL37x1 or Towne-BAC infected live cell cultures (Fig. 10). By day 5, foci with brightly stained GFP+ debris was observed in cultures infected with either virus (Fig. 10E–F), although fragmentation was rare in wt virus-infected cultures at this time. The SLCK staining pattern was distinct in ΔUL37x1-infected cells and included bright SLCK+ debris (Fig. 10A) that was distinguishable from Towne-BAC infected cells by differences in the amount of staining as well as the size and distribution of debris (Fig. 10A–B). By 8 days after infection, most ΔUL37x1-infected cells in each plaque were brightly fluorescent (Fig. 10C) whereas cells infected with wt virus (Fig. 10D) showed only SLCK+ debris. SLCK staining patterns did not appear to be mitochondrial at any time in either virus infection. These patterns were distinct from HtrA2/Omi (Fig. 6C), suggesting that SLCK labeling detected serine proteases in addition to HtrA2/Omi. Addition of 0.1 or 1 mM TLCK reduced but did not eliminate SLCK binding to mutant virus-infected cells, consistent with the induction of serine proteases (Fig. S11). Overall, >50% of GFP+ cells in ΔUL37x1 plaques also labeled with SLCK. SLCK staining was reduced to ≤30% by addition of 100 µM TLCK. Thus, SLCK revealed a higher level of protease activation in CMV infected cells that were susceptible to premature cmvPCD. This data suggests that vMIA may control a broader serine protease-dependent death pathway by counteracting mitochondrial HtrA2/Omi during viral infection.10.1371/journal.ppat.1000063.g010Figure 10SLCK localization within infected cells.Images of fluorescent serine protease substrate SLCK localization (A–D) and GFP fluorescence (E–H) in ΔUL37x1 (A, C, E, G) or Towne-BAC (B, D, F, H) foci undergoing fragmentation at 120 h (A–B, E–F) and 192 h (C–D, G–H) postinfection (MOI of 0.001). Arrows indicate SLCK-bound debris. Original magnification ×400 (A–H).DiscussionCMV replicates in the nucleus, matures in the cytoplasm and is released into the surrounding medium or adjacent cells over the course of a 7 to 10 day replication cycle [1]. Host cells are dramatically reprogrammed for production of progeny virus until death occurs via a process that begins late in CMV infection, associated with late gene expression that drives CPE and cell cycle dysregulation [73]–[76]. In an effort to define viral and cellular contributions to morphological and biochemical events that terminate CMV infection, we have discovered the key role of mitochondrial HtrA2/Omi and a novel cell death pathway. This cellular serine protease appears to be responsible for induction of cmvPCD following a pathway that is held in balance by the viral cell death suppressor, vMIA. vMIA resides in the mitochondrion where it is a potent suppressor of cytochrome c release, thereby preventing activation of executioner caspases during apoptosis [2]–[4]. In addition to suppression of apoptosis, vMIA carries out a distinct and nonoverlapping role suppressing death induced by HtrA2/Omi during the late phase of viral infection. This cmvPCD pathway is triggered only in the context of infection. Late phase infected cell events promote cell fragmentation together with collapse, shrinkage, and loss of nuclei in a pathway that is dependent on HtrA2/Omi protease activity and associated with the activation of cytoplasmic serine proteases that may act as executioners. HtrA2/Omi levels rise before induction of death, consistent with a central role of this protease in initiation of cmvPCD. Suppression of this death pathway, like suppression of apoptosis, is associated with global disruption of mitochondrial networks by vMIA. Unlike apoptosis, however, cmvPCD apparently does not require cytochrome c release from mitochondria to trigger downstream events. Further, HtrA2/Omi remains mitochondrial late during infection suggesting death may be initiated by the activity of the intramitochondrial protease, which raises an interesting question as to how transduction of the death signal occurs. Our data reveal a pathway that is triggered by high intramitochondrial HtrA2/Omi protease and controlled by vMIA.Although vMIA-mutant virus undergoes premature cmvPCD, the fragmentation process is similar to cmvPCD in wt virus-infected cells. The difference appears to be in timing of cell death. vMIA delays death for several days beyond the initiating trigger which is coincident with the late phase of replication. Although induction of HtrA2/Omi is independent of vMIA, the impact of induction appears to be the target of vMIA function at the mitochondria where both reside. Suppression of cmvPCD benefits the virus by extending the period of virus production by infected cells [5], although cultured fibroblasts show only slight reduction in yield and cell-to-cell transmission in the absence of vMIA. A prolonged period of virus production increases the amount of virus released cell-free and potentially benefits transmission in natural settings. A delay in fragmentation would also delay phagocytosis and clearance of virus-infected cells [77]. HtrA2/Omi-dependent death may be viewed as an intrinsic host antiviral process analogous to apoptosis. vMIA control of HtrA2/Omi-mediated death is analogous to control of apoptosis, as both appear to be independent cell-intrinsic mechanisms of pathogen control. Importantly, vMIA appears to provide concurrent protection from both pathways.vMIA disruption of reticular networks and organization of mitochondria [22] is independent of HtrA2/Omi accumulation within mitochondria, but does correlate with cell death suppression activity. Thus, ΔUL37x1-mutant virus-infection preserves mitochondrial networks throughout infection, during HtrA2/Omi accumulation and initiation of premature cmvPCD. In contrast, wt virus infected cells support the same accumulation of HtrA2/Omi and a vMIA-driven disruption of mitochondrial networks but survive. The correlation between this vMIA-dependent disruption and cell death protection suggests that punctate mitochondria may be more resistant to the stress induced by late phase events. Reticular mitochondria are known to rapidly disseminate Ca++ or ATP signals; whereas, punctate mitochondria have slower responses to changes in intracellular mediators [78]. Additional experiments will be needed to understand the mechanism underlying resistance of punctate mitochondria to death, whether mediated via caspases or HtrA2/Omi.Emerging evidence suggests vMIA, viral strain differences, and cellular factors contribute to the control of mitochondria and death. Thus, AD-BAC kills cells earlier [6] and disrupts mitochondrial networks by 24 h postinfection [22] whereas Towne-BAC disruption occurs later, by 48 h postinfection and cells die later. vMIA associates with the outer mitochondrial membrane within 24 h [79],[80]. AD-BAC (or its parent AD169varATCC) depends upon vMIA to suppress caspase-dependent apoptosis that develops by 48 to 72 h postinfection. Towne-BAC (or its parent TownevarATCC) depends on vMIA to suppress caspase-independent, HtrA2-dependent cell death that develops by 72 to 96 h postinfection. It remains to be seen whether vMIA suppresses both pathways in cells infected with strains like AD-BAC. Accumulation of HtrA2/Omi occurs in other viral strains (McCormick, unpublished), underscoring the overall importance of the process described here. There are many potential factors contributing to qualitative or quantitative differences in the way characterized viral strains initiate and control death, with apoptosis apparently predominating in some settings and HtrA2-mediated death predominating in others. We focused here on dissecting the novel death pathway in Towne-BAC-infected cells, to characterize a novel HtrA2/Omi pathway that is independent of apoptosis.cmvPCD may be influenced by or even associated with a number of additional modulatory effects of this virus that impact late times of infection, including dysregulation of the cell cycle [73]–[76], disruption of p53 activation [81], DNA damage response [82],[83] and unfolded protein response [84] that all remain incompletely understood. vMIA may reduce ATP levels during infection as it does in established cell lines. Although suggested to control late CPE in AD-BAC derivatives [85] vMIA has no impact on development of CPE in Towne-BAC derivatives. Any vMIA-specific reduction in ATP levels is likely highly coordinated with other viral processes contributing to late CPE. CMVs encode multiple factors that target mitochondria [86],[87], regulate expression of mitochondrial proteins [75] and even stimulate mitochondrial DNA synthesis [88] suggesting viral control of mitochondria functions is complex. The vMIA-specific impact on ATP levels as related to HtrA2/Omi remains unknown but may certainly be a feature of control. As an event that occurs very late in replication, cmvPCD is crucial to sustaining viral infection in individual cells. Our observations that either mutation of vMIA or premature overexpression of HtrA2/Omi levels dramatically alters the timing of death indicate that these two may balance each another in controlling cmvPCD.Previously, pharmacological inhibitor and overexpression studies have implicated HtrA2/Omi as a regulator of death [35],[37],[40],[89],[90]. Genetic studies have suggested this protease functions primarily to ensure normal mitochondrial homeostasis [39],[91],[92], perhaps controlling protein quality and cellular stress responses [34] similar to the related bacterial protease HtrA [41]–[43]. The role of HtrA2/Omi in caspase-independent cell death has not previously been studied in detail, although the truncated, active form of HtrA2 drives death when released from mitochondria or expressed directly in the cytoplasm [35],[37],[40],[90],[93]. We have shown that the active form drives death specifically and only in CMV-infected cells, which we correlate with the fact that the enzyme remains mitochondrial throughout CMV infection. CMV infection is a unique setting that has unveiled a direct role for HtrA2/Omi in a caspase-independent cell death pathway analogous to apoptosis.vMIA controls the programmed death of infected cells after a week or more of replication, following a period of persistent virus production. CMV infects many cell types in addition to HFs, and given that the timing of replication varies with cell type, vMIA control of HtrA2/Omi-dependent death may be critical in other cell types or in natural infection of the human host. Given the many functions that CMV has evolved to manage the virus:host standoff, we speculate that viral control of cmvPCD represents a benefit to the virus, potentially allowing infected cells to avoid sending alarm signals. Other examples of viral proteins acting together to control the type of cell death that follows replication can be identified. Thus, the adenovirus death protein, ADP, functions in the presence of E1B-19k, the viral Bcl-2 protein, and both contribute to the type of death that terminates infection [94]–[96]. Caspase-dependent apoptosis is itself a cell-intrinsic pathogen clearance process, minimizing inflammation and pathology while alarming the immune system to initiate cellular responses [77]. CMV-encoded cell death suppressors provide a means of evading cell death directed by host cell intrinsic, innate, and adaptive responses [97]. The benefit of controlling the mechanism and timing of cell death includes persistence, as well as the interface of virus-infected cells with the host immune system. In the host, cmvPCD may provide for greater success in attaining a foothold without evoking clearance. The presence of vMIA-like functions in other cytomegaloviruses [5],[17] as well as the broad distribution of mitochondrial cell death suppressors in other viruses suggests this novel serine protease pathway may occur in other biological settings.Materials and MethodsAccession numbersThe HtrA2 protease has MEROPS accession number S01.278 and the I.M.A.G.E. consortium clone obtained for these studies was identical in sequence to NCBI accession ID BC0000096. The vMIA [2] used to complement and repair ΔUL37x1 was obtained from AD169varATCC genomic DNA; NCBI accession ID X17403. The sequence of Towne-BAC was deposited to NCBI [8]; accession ID AY315197.Cells and virusesHuman fibroblasts (HFs), vMIA-HFs, vMIAmut-HFs, and HeLas were cultured as previously described [5]. Viruses derived from the BACmid clones Towne-BAC and ΔUL37x1 [8] were maintained as DNA clones in E. coli or on complementing vMIA-HFs prior to experiments. AD169varATCC was maintained as previously described [22].Generation of ΔUL37x1 rescue virus RC2707The kanamycin selection cassette in ΔUL37x1 was replaced with UL37x1 sequence derived from AD169varATCC to generate RC2707. Transfection of pON2707 [5] into HFs was followed by infection with ΔUL37x1 virus. Plaques that included cell death at a frequency similar to Towne-BAC virus [5] were isolated for further analysis. Sequencing of viral DNA from Towne-BAC and two independently derived isolates (ΔUL37x1R1, ΔUL37x1R2) confirmed replacement of the selection marker in ΔUL37x1 with UL37x1 nucleotide sequence identical to pON2707 and AD169varATCC while the control, Towne-BAC, was identical to the expected sequence [8],[98]. Expression of vMIA was confirmed by immunoblot analysis.PlasmidsThe HtrA2/Omi expression plasmid, pON601, was derived by restriction of the I.M.A.G.E. cDNA (HtraA2 clone #5344667, ATCC, Manassas, VA) with BsrGI, followed by removal of the single-stranded overhangs with Klenow DNA polymerase, and restriction with XhoI. The HtrA2/Omi encoding fragment was ligated to EcoRV and XhoI restricted pcDNA3.1+ (Invitrogen, San Diego, CA). The HtrA2S306A expression plasmid, pON602, was generated by PCR-site directed mutagenesis of the HtrA2 ORF to introduce the S306A mutation and a novel NaeI restriction enzyme site and utilized the mutagenic primer 5′-CTATTGATTTTGGAAACGCCGGCGGTCCCCTGGTTAAC-3′. Both clones were sequenced to confirm expected results. The vMIA and GFP expression clones and retroviral constructs used in these experiments were reported previously [2],[5],[18].Immunoblotting, immunofluorescence, and MitoTracker Red staining assaysImmunodetection employed mouse monoclonal antibodies to c-myc epitope (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), HtrA2/Omi (MAB1458; R&D Systems, Inc, Minneapolis, MN), cytochrome c (Clone 7H8.2C12, BD Pharmingen, San Jose, CA), β-actin (AC-74, Sigma, St. Louis, MO), golgin-97 (CDF4; Molecular Probes, Eugene, OR), mitochondrial heat shock protein 70 (mtHSP70) (a gift from Susan Pierce, Northwestern University), viral nuclear antigens IE1p72 and IE2p86 (MAB 810, Chemicon, Temeculah, CA), ICP36 (CH16) and pp28 (CH19) (both from Virusys Corporation, Randallstown, MD) or rabbit polyclonal antiserum to native vMIA [2] and peroxidase-conjugated horse anti-mouse IgG or goat anti-rabbit IgG, Texas Red-conjugated horse anti-mouse IgG (all from Vector, Burlingame, Calif.), or AlexaFluor 350-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR). Immunoblot analysis of total protein from infected cells and immunofluorescence assays followed previously described methods [22]. MitoTracker Red CMXRos (Molecular Probes, Eugene, OR) staining of mitochondria followed previously described methods [22].Detection of cmvPCD, impact of replication and protease inhibitors on death, viral yield, and BACmid transfectionsTo assess morphological changes in infected cells and nuclei, cells grown on coverslips and infected for varying periods of time were fixed with 3.7% formaldehyde, permeabilized with Triton X-100 (EMD Biosciences, Darmstadt, Germany), stained with Hoechst 33258 (AnaSpec, San Jose, CA), and processed for microscopic evaluation as previously described [22]. Some cultures were stained with ethidium homodimer 1 (Molecular Probes, Eugene, OR), as previously described [5] to assess virus-induced cell death.Images from live cell cultures were obtained as previously described [5] or utilized Simple PCI software, a Retiga Exi digital camera, and a Leica DM IRB microscope. Imaging of cultures grown on coverslip employed an AxioCam MRc5 camera attached to a Zeiss Axio Imager.A1 and AxioVision Release 4.5 software.Replication inhibitors included phosphonoformate (PFA Sigma, St. Louis, Mo) dissolved in water and 2-bromo-5,6-dichlorobenzimidazole (BDCRB from L. B. Townsend, University of Michigan) dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO). Protease inhibitors included TLCK, N-alpha-p-tosyl-L-lysine chloromethyl ketone, (Sigma, St. Louis, MO) in water, and UCF-101 or zVAD.fmk (both from Calbiochem, La Jolla, CA) dissolved in DMSO. Inhibitors were added by replacing culture medium with medium containing inhibitor while control medium included the appropriate solvent (DMSO) or no addition. Morphology and presence of viral nuclear antigens IE1p72 and IE2p86 were assessed as described above and viral yield was determined from total virus recovered on day 7 from supernatant and sonicated cells [5]. DMSO does not impact CMV death or CMV replication levels at the concentrations used (≤0.1%)[5].Transfections of BACmid DNA have been described [5]. GFP-positive (GFP+) cells and GFP-positive foci (>2 GFP+ cell) were evaluated by live cell microscopy 2–10 days post transfection. Viral presence was confirmed by immunodection of viral nuclear antigens IE1p72 and IE2p86 and some experiments utilized a plasmid encoding GFP [5] for detection of transfected cells. Reported results were obtained from multiple DNA preparations.Apoptosis and viability assaysConditions for induced apoptosis of ADvarATCC infections and vMIA-dependent survival following transfection of HeLa have been described [2],[64]. Cell numbers were determined following Hoechst stain of surviving cells (HeLas) or following immunodetection of viral nuclear antigens IE1p72 and IE2p86 (ADvarATCC), comparing to untreated controls.To assess the impact of HtrA2/Omi on HFs, GFP expression plasmid was cotransfected with control DNA (vector) or HtrA2/Omi or HtrA2S306A expression plasmids. By 48 h cells had recovered and were confluent. Images obtained of GFP fluorescence at 24 h intervals between 48–120 h post transfection were evaluated for numbers of GFP+ cells from 12 microscopic fields per day. Mean % survival (±standard deviation) was calculated from numbers of GFP+ cells compared to those at 48 h.SLCK labelingSulforhodamine 101-leucine chloromethyl ketone, SLCK, (Immunochemistry Technologies, LLC, Bloomington, MN) was suspended in DMSO (Sigma, St. Louis, MO). For labeling, 2.5 µM SLCK was applied in the presence or absence of TLCK to live cultures for 30 minutes prior to fixation and imaging as described above.Supporting InformationFigure S1Towne-BAC cmvPCD occurs after viral release. Examples of single infected cells (A, B) or foci (C, D) remaining intact and live (A, C) or exhibiting fragmentation and death (cmvPCD) (B, D) are shown. Original magnification ×400. (E) Percentages of single infected cells (patterns A+B) and foci (patterns C+D) for ΔUL37x1 and Towne-BAC. (F) Percentages of live, nonfragmented single cells or foci at 72, 96, or 120 h postinfection (hpi) with ΔUL37x1 or Towne-BAC. (G) Percentages of dead single cells or foci at 72, 96, or 120 hpi with ΔUL37x1 or Towne-BAC. A total of 400 infected cells/foci per virus were evaluated at each time for the experiment depicted in panels E–G following infection at MOI 0.0001. The mean±sd is depicted in all figures, except where indicated.(1.84 MB TIF)Click here for additional data file.Figure S2Nuclear and cytoplasmic inclusions indicate similar late cytopathic effects in Towne-BAC and ΔUL37x1 infections. Representative fluorescent images of nuclear and cytoplasmic inclusion proteins ppUL44 (A, C) and ppUL28 (E, I) (red), respectively, in ΔUL37x1 (A–B, E–H) and Towne-BAC (C–D, I–L) infected cells (MOI of 0.001). (GFP = green, Hoechst = blue) Original magnification ×1000.(6.21 MB TIF)Click here for additional data file.Figure S3cmvPCD intermediates do not stain with ethidium homodimer. Representative fluorescent images of GFP (A, E, I, M, Q) (green), ethidium homodimer (B, F, J, N, R, U) (red), Hoechst (C, G, K, O, S, V) (blue), and merged images (D, H, L, P, T, W) in ΔUL37x1 infected intact (A–D) or fragmenting (E–T) cells stained prior to fixation, and in controls fixed with methanol prior to labeling (U, V, W). Original magnification ×1000.(2.45 MB TIF)Click here for additional data file.Figure S4Towne-BAC death intermediates. Representative fluorescent images of Towne-BAC infected cells (MOI of 0.01) showing cmvPCD (A–D) at 168 h postinfection (hpi). Original magnification ×400.(1.99 MB TIF)Click here for additional data file.Figure S5HtrA2/Omi expression following CMV infection. Immunoblot analyses of HtrA2/Omi in lysates of mock-infected HF (M) or HF infected (MOI of 3) with Towne-BAC or ΔUL37x1 for 24, 48, 72, 96, or 120 h. Control cell lysates from HeLa cells transfected with HtrA2/Omi expression plasmid (+) or control plasmid (−). Immunoblot detection of HtrA2, β-actin and IE1 are shown.(0.50 MB TIF)Click here for additional data file.Figure S6Mitochondria are reticular in ΔUL37x1 and punctate in Towne-BAC infections. Representative fluorescent images of mitochondria HSP70 (mtHSP70) (A, E), golgin 97 (I, M), HtrA2/Omi (Q, U), and cytochrome c (Y, c) (blue), and MitoTracker Red stain of mitochondria (B, F, J, N, R, V, Z, d) (red), and GFP fluorescence (C, G, K, O, S, W, a, e) (green) and merged images (D, H, L, P, T, X, b, f) at 72 h postinfection (MOI of 0.001) with ΔUL37x1 (A–D, I–L, Q–T, Y–b) or Towne-BAC (E–H, M–P, U–X, c–f). Original magnification ×1000.(3.53 MB DOC)Click here for additional data file.Figure S7Reticular cytochrome c pattern maintained until after initiation of cmvPCD. Representative fluorescent images of cytochrome c (A, D, G, J, M), GFP fluorescence (B, E, H, K N) and Hoechst (C, F, I, L, O) at 96 h postinfection (MOI of 0.001) in fragmented ΔUL37x1 infected cells. Arrow in B indicates fragments of GFP+ cell. Original magnification ×1000.(4.19 MB TIF)Click here for additional data file.Figure S8HtrA2 overexpression in HeLa cells does not induce death. GFP fluorescence following cotransfection of HF with GFP expression plasmid together with empty vector, HtrA2/Omi or HtrA2S306A expression plasmids at 24, 48, and 72 h posttransfection. Original magnification x40.(2.56 MB TIF)Click here for additional data file.Figure S9HtrA2 reduces plaque formation following a decrease in viability of infected cells evident by 72 h postinfection. Combined numbers of GFP+ cells and foci following cotransfection of Towne-BAC DNA (500 ng) with 800 ng vector or HtrA2/Omi expression plasmid.(0.16 MB TIF)Click here for additional data file.Figure S10vMIA impact on cmvPCD. (A) Total number of ΔUL37x1 plaques on day 10 following infection at MOI 0.0001 of HF, vMIAmut-HF, or vMIA-HF (B) Total viral yield following infection of HF by ΔUL37x1, Towne-BAC, ΔUL37x1R1, or ΔUL37x1R2 for 10 days at MOI 0.0001. (C–H) Mitochondria in ΔUL37x1R1 and ΔUL37x1R2 infected cells at 72 h postinfection. MitoTracker Red stain (C–F) (red), GFP fluorescence (D, G), and the merged images. Original magnification ×1000.(4.66 MB TIF)Click here for additional data file.Figure S11Serine proteases labeled with SLCK and impact of TLCK added as inhibitor. Representative images of fluorescent serine protease substrate SLCK localization (A–C) and GFP fluorescence (D–F) in ΔUL37x1 foci undergoing fragmentation on day 8 postinfection (MOI of 0.001) in the presence of no addition (A, D), 100 µM TLCK (B, E), or 1 mM TLCK (C, F) Original magnification ×200.(4.49 MB TIF)Click here for additional data file.\n\nREFERENCES:\n1. 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+}

batch_10/PMC2528008.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2528008",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528008\nAUTHORS: Katrina D Bello, Nahal Goharpey, Sheila G Crewther, David P Crewther\n\nABSTRACT:\nBackgroundAssessment of 'potential intellectual ability' of children with severe intellectual disability (ID) is limited, as current tests designed for normal children do not maintain their interest. Thus a manual puzzle version of the Raven's Coloured Progressive Matrices (RCPM) was devised to appeal to the attentional and sensory preferences and language limitations of children with ID. It was hypothesized that performance on the book and manual puzzle forms would not differ for typically developing children but that children with ID would perform better on the puzzle form.MethodsThe first study assessed the validity of this puzzle form of the RCPM for 76 typically developing children in a test-retest crossover design, with a 3 week interval between tests. A second study tested performance and completion rate for the puzzle form compared to the book form in a sample of 164 children with ID.ResultsIn the first study, no significant difference was found between performance on the puzzle and book forms in typically developing children, irrespective of the order of completion. The second study demonstrated a significantly higher performance and completion rate for the puzzle form compared to the book form in the ID population.ConclusionSimilar performance on book and puzzle forms of the RCPM by typically developing children suggests that both forms measure the same construct. These findings suggest that the puzzle form does not require greater cognitive ability but demands sensory-motor attention and limits distraction in children with severe ID. Thus, we suggest the puzzle form of the RCPM is a more reliable measure of the non-verbal mentation of children with severe ID than the book form.\n\nBODY:\nBackgroundIntellectual disability (ID) affects 1.25% of the Australian population [1] and is defined according to the ICD-10 criteria as ongoing difficulties in age appropriate functioning and below age average cognitive performance as demonstrated by a score of two standard deviations below the mean on standardized intelligence tests. However, standardized intelligence tests such as the WISC-IV are often limited in their assessment of children with severe intellectual disability (ID) who are often unable to stay on task for the lengthy administration of the test, or handle its heavy reliance on language skills [2-4] and lack of ability to motivate [5]. Thus, to produce a valid measure of cognitive ability for children with severe ID, testing procedures must accommodate their profound deficits in communication, attention and social skills [6-10]. Such procedures are necessary and important to facilitate the most appropriate educational placement to enhance their educational and learning potential. We suggest that the Raven's Coloured Progressive Matrices Test (RCPM [11]) is a potentially more suitable alternative to tests like the WISC as it is an untimed non-verbal measure of reasoning ability [3,12,13]. This is supported by a recent study by Dawson, Soulières, Gernsbacher and Mottron [14], which showed that the WISC-III underestimates intelligence in children with ASD. They found that scores of 38 children with ASD were on average 30 percentile points higher on the Raven's Progressive Matrices (RPM) than their scores on the WISC-III, whereas no such difference was found for typically developing (TD) children. The RCPM consists of 36 coloured multiple choice matrices (although colour is irrelevant to the completion of the task), organized in three increasingly complex sets [3,11-15]. It is being utilized increasingly with children with severe ID, including those with Autism Spectrum Disorder (ASD) [5,16] in research settings to control for non-verbal mentation [13,17,18] and in educational settings to determine the level of functioning and treatment progress as part of a battery of tests [19,20].Despite it being a better indicator of non verbal cognitive ability than the WISC III, many children with severe ID still show difficulties in completing the RCPM. Clark and Rutter [16] found that motivation and associated disruptive behaviours such as task avoidance, self-stimulation and escape behaviours in children with lower functioning ASD, hindered test performance on the RCPM. Techniques adopted to maintain motivation (e.g. lowering task difficulty to increase success rate in low scoring children) led to better performance, which suggests that the task itself is not sufficiently engaging of attention for children with impaired intellectual functioning. The standard book form of the RCPM also requires the child to point to their chosen pattern, which is a problem as pointing is one of several delayed social communication skills observed in many children with ID, particularly ASD [21].To enhance compliance in cognitively less able clinical groups, Raven produced a board form of the RCPM [15] where each item, presented on a wooden board, can be completed with the correct placement of movable pieces. Raven et al. [15] claim that the board form is a consistent, reliable and psychologically valid estimate of reasoning ability, with a test retest reliability of approximately r = 0.80. However, although past studies [16,22-24] have utilized the board form, the study details are not available and, evidence of its validity is limited. Furthermore its heavy inflexible wooden design is often unsuitable for use for children with severe ID. Carlson and Weidl used a test-retest design to show that the board form produced better performance than the book form in typically developing children [22] and children with ID [23]. However, because they allowed for trial and error in the completion of the board form, it is unclear whether the better performance on the board form was due to increased opportunity for self-correction or the nature of the board form itself. The board form is also limited as the moveable pieces are easily disarranged when in use and administration of 36 separate board pieces is quite time consuming [15]. Such task characteristics do not encourage sustained attention and motivation in children with severe ID.In line with the merits of the board form and considering its administrative inflexibility we have designed a puzzle version as an alternate form of the RCPM specifically designed to encourage greater sensory attention and motivation, increase task comprehension and consequently limit other disruptive behaviours in order to obtain a more valid measure of reasoning ability in children with ID. This new form resembles a jigsaw puzzle and therefore minimizes verbal task instructions for children with severe ID [25]. It is also conceptually like the board form in that participants must physically remove pieces, however, our puzzle form utilizes a cardboard and Velcro™ system to allow the children to simply grasp and easily remove their chosen piece and place it in the gap of the larger pattern. Unlike the board form, the puzzle form is presented in a folder with each item displayed individually on one page and each piece secured with Velcro to minimize weight, distractions and ease and time of administration. Another advantage of the puzzle form is that grasping the pieces maintains attention better than the requirement of pointing, as in the book form. This is consistent with the idea that grasping requires more brain activation than visual recognition alone [26]. Grasping requires processing of spatial location, in addition to form, orientation and size [27] and serves to draw attention to the object, which maintains attention on the task. Motor engagement with the pieces and placement in the appropriate area provides immediate feedback and requires more attentional resources. Kaplan et al. [28] showed that people with ID receiving sensory input from different pieces of equipment, showed less aggression and self-stimulatory behaviour and more task completion. This effect was also generalized to subsequent tasks, which supports the effect of tactile stimulation in increasing task engagement in people with ID. Motor engagement is particularly important in children with severe ID and children with ASD who are less motivated by social reinforcement [29] perhaps due to they failure to orient to and engage with the affective expressions of others [28,30,31]. Doussard-Roosevelt, Joe, Bazhenova and Porges [32] found that children with ASD were more engaged when their mothers physically and non-verbally demonstrated an object to them than when she verbally described the object to them.Thus the aims of these studies were in Study 1, to test the validity of performance of typically developing (TD) on the puzzle form of the RCPM by comparing it to the standard book form; and in Study 2, to examine overall performance and completion rate of the puzzle and book form in children with idiopathic ID, Down Syndrome (DS) or ASD to establish the potential applicability of this alternative puzzle form to children with severe ID. We hypothesized that, in Study 1, TD children would show comparable performance in the book and puzzle form of the RCPM, irrespective of which form was completed first on a counterbalanced cross over design over a three week period. We also hypothesized that, in Study 2, children with severe ID, whether ID, DS or ASD, who completed the puzzle form, would show a higher performance rate than children who completed the book form, irrespective of clinical group.Study 1: Comparison of the standard and puzzle forms for the validation of the puzzle form of the RCPMMethodsParticipantsSeventy-six typically developing (TD) children attending a mainstream primary school within the Catholic education system in the northeastern suburbs of Melbourne, Australia, participated in the current study. Participants were aged between 5 and 11 years (M = 8.57 years, SD = 2.06 years), 40 of whom were male, and 36 were female. Participants were required to speak English as a primary language and fall within the middle range for socio-economic status backgrounds. Participants had no known neurological intellectual disabilities and were screened for hearing problems and for normal or corrected to normal vision. Participants were randomly assigned to a group who complete the book form first or another group who completed the puzzle form first. Table 1 shows the chronological age and RCPM score of each group. As can be seen, the groups were closely matched and were not significantly different for age, t(74) = 0.45, p > .05.Table 1Means and standard deviations for age for children who were all randomly assigned to one of two groups that either completed the standard book form first or the puzzle form first.GroupNAge (years) (SD)RCPM score (SD)Total768.6 (2.1)25.6 (6.1)Book form388.7 (2.1)25.5 (5.7)Puzzle form388.4 (2.1)25.8 (6.7)Ethics approval for studies 1 and 2 was obtained from the Swinburne University of Technology and La Trobe University Human Ethics Committees. Permission to conduct testing in the school was obtained from the Catholic Education Office in Victoria, and the Principal of the School. Individual parental or guardian consent for each child was required prior to testing and all children were free to withdraw from testing at any time.MaterialsThe RCPM is comprised of 36 items divided into three subsets of 12 items (Sets A, Ab, and B). Each item consists of a different coloured pattern with six possible pieces available to fill the \"missing\" location required to complete the pattern. The participant's task was to deduce the theme of relations expressed among the designs and choose the missing figure from among the alternative set of six. The original book form displayed each item on a page in a booklet. The alternative puzzle version was the same size and colour as the book form, but differed in that each of the alternative patterns could be removed and physically attached to the missing place on the matrix through the use of a Velcro system.ProcedureThe standard administration procedure as prescribed by Raven et al [9], was used for the original book form, with trained clinicians administering both book and puzzle forms individually to each child [11,15], within the school setting. As suggested by Raven et al. no time limit was assigned for either task. Participants were required to select a piece from six alternatives that completed the pattern for each item by either pointing to their chosen response in the book form or by removing their chosen response and placing it in the missing section of the matrix in the puzzle form. Participants were asked to do this using the verbal instruction \"find missing\". This very simple, clear and short verbal instruction was chosen to ensure that it could be successfully used with children with ID who have limited receptive language. Participants were required to select a piece their responses by pointing to Item one of the standard and puzzle versions served as a practice trial, where incorrect responses were corrected and no further assistance or verbal reward was given during performance and completion of the task. Performance on the RCPM was calculated according to the number of items correct, and unattempted items were classified as incorrect. Inclusion criteria required children to attempt at least one full set of 12 items. Children attempting less than this, were excluded from further analyses.In Study 1, the TD children were randomly assigned to two groups where one group attempted the book form first while the other half attempted the puzzle form. The alternate form of the RCPM was again administered after three weeks. To minimize the impact of maturation in learning and memory or practice effects on performance a three-week interval between the puzzle and book form was used [13,33].Data AnalysesTo validate the puzzle form, the performance of children who completed the standard book form first was compared to the performance of children who completed the puzzle form first using an independent samples t-test. A comparison of the two versions using a cross-over design was then used to examine the puzzle version performance over time, and to show that it matters little to overall performance of TD children, which form of the test was performed first. Previous test-retest studies using only the book form of the RCPM were conducted three weeks apart and reported correlations of Pearson's r = 0.80 [13,34,35]. As an alternative measure to Pearson's r, interclass correlation coefficient (ICC) [31] and coefficient of variation of measurement error (CVME) [31] were also calculated for an indication of degree of relatedness and percentage of variation respectively, between scores from the first and second test occasions.ResultsData were initially screened for outliers and any violations of the assumptions of normality, homogeneity of variance, and sphericity. No outliers or violations of assumptions in the data were detected.Comparison between performance on standard and puzzle formsTable 1 shows the RCPM means and standard deviations for the TD participants who completed the original book form and the group who completed the puzzle version. It can be observed from Table 1 that the mean score for each group was similar and an independent samples t-test showed no significant difference in RCPM score between children who completed the original book form and children who completed the novel puzzle form, t (74) = -0.22, p > .05.Cross-over designAs displayed in Figure 1, the mean raw performance score for the first attempt was lower than for the second attempt irrespective of which version was completed first. A repeated measures ANOVA found this to be a significant effect, F(1, 74) = 8.62, p < .05. No significant interaction effect F(1, 74) = 0.14, p > .05 was found.Figure 1Mean and standard error of RCPM score for typically developing participants who completed the original book form first and those who completed the puzzle version first.As presented in Table 2, a high correlation, r = 0.85, p < .01, was found between first and second attempt regardless of the form. The correlation between the first and second attempt for participants who completed the puzzle form first was higher, r = 0.93, p < .01, than for participants who completed the standard form first, r = 0.76, p < .01. This pattern was also observed with the ICC and CVME measures in that respectively, the degree of relatedness between first and second test occasions was greater for those who completed the puzzle form first compared to those who completed the book form first; and the percentage of variation between scores from the first and second test occasions was less in those who completed the puzzle form first compared to those who completed the standard form first.Table 2Correlation coefficients Pearson's r, interclass correlation coefficient (ICC), and coefficient of variation of measurement error (CVME) values for RCPM score for first and second attempt for children who completed the book first and children who completed the puzzle first.GroupNRICCCVMETotal760.850.827.22%Book first380.760.747.89%Puzzle first380.930.886.70%A large test-retest reliability score (r = 0.85, p < .01) was found between the standard book form and the puzzle version, in TD children. This correlation is comparable to past studies solely examining the RCPM book form using a similar time frame of three weeks [32-34]. The findings suggest that the puzzle form is as useful as the standard book form of the RCPM in measuring nonverbal mentation in typically developing children.In summary, the findings of Study 1 support the hypothesis that the book and the puzzle forms are measuring similar constructs in TD children. This suggests that the puzzle form, can be used with children with severe ID and potentially enhance performance and completion rate whilst still measuring the same constructs as the book form. Study 2 was conducted to examine the use of the puzzle form of the RCPM to measure non-verbal mentation in children with ID to evaluate the hypothesis that the puzzle form maintains attention in such children.Study 2: The puzzle form of the RCPM to measure non-verbal mentation in children with Intellectual DisabilityMethodOne hundred and eighty-nine children with ASD, Down Syndrome (DS) or idiopathic ID, recruited from specialist schools in metropolitan Melbourne, Australia, were originally administered the book or puzzle form, but 25 participants were unable to complete a minimum of 12 items and were therefore excluded from further analyses. Table 3 shows the means and standard deviations for chronological age for the remaining 164 children, divided into clinical groups. Inclusion criteria from Study 1 were also used in Study 2. Participants were randomly assigned to be administered either the book form or puzzle form. The puzzle and book form were administered as detailed in Study 1.Table 3Means and standard deviations for age for each group of children with Autism Spectrum Disorder (ASD), Down Syndrome (DS), and idiopathic intellectual disability (ID).GroupNChronological Age (years) (SD)Total16410.7 (3.9)ASD1019.7 (3.5)DS2011.8 (3.7)ID4310.6 (3.5)Given that the data from this study were not normally distributed, non-parametric testing was used for all analyses.ResultsThe RCPM means and standard errors for each ID group administered the book and puzzle forms are shown in Figure 2. A Kruskal-Wallis test showed no significant differences in RCPM score between the clinical groups, H (2) = 3.26, p > .05. A Mann-Whitney test showed a significant difference in RCPM score between performance on the book and puzzle form regardless of clinical group, Z = -5.54, p < .05. When each clinical group is examined separately, the ASD group participants who were administered the puzzle form performed significantly better than those who were administered the book form (Z = -3.99, p < .05) and the ID group (Z = -3.31, p < .05) but not the DS group (Z = -1.60, p > .05).Figure 2Mean RCPM score of children with ASD (n = 101), DS (n = 20), and ID (n = 43) who completed the book form or the puzzle form of the RCPM.While the inter-group RCPM performance and hence mentation age was not significantly different, there was a significant difference in age between the three clinical groups F (2, 161) = 13.20, p < .05, with the mean age of the ASD group significantly less than the DS and ID groups. However, the age difference between children administered the puzzle and book form was not significantly different for each clinical group (ASD t (99) = -1.20, p > .05; DS t (18) = -0.78, p > .05; ID t (41) = 0.44, p > .05).As displayed in Figure 3, completion rate for the puzzle form (76.2%) was greater than for the book form (40%), regardless of clinical group. A Mann-Whitney test showed a significant difference in RCPM score between children who were able to complete the RCPM test and children who attempted at least 12 items but were unable to complete the task, regardless of which form they were administered, Z = -10.55, p < .05. Of those children who were unable to complete the book form, 55% of children with ASD, 68% of children with DS, and 67% of children with ID were able to complete the puzzle form. The results suggest that the use of the puzzle form as compared to the book from of the RCPM has resulted in better task performance and completion rate for all clinical groups.Figure 3Percentage of children with ASD, DS, and ID who completed the book or the puzzle form of the RCPM.To deal with the potential confound of completion rate, further analyses were performed only on participants who completed the puzzle or book form. A Mann-Whitney test showed that the participants who completed the puzzle form performed significantly better than those who completed the book form, Z = -2.89, p < .05. From Figure 4, it can be seen that in each clinical group, those who completed the puzzle form performed better than those who completed the book form, but only the ASD group showed this difference to be statistically significant (Z = -2.52, p < .05), but not the DS (Z = -0.19, p > .05) and ID (Z = -1.61, p < .05) groups.Figure 4Mean RCPM score of children with ASD, DS, and ID who were able to complete the book form or the puzzle form.DiscussionThe finding from Study 1 of no difference between the performance of TD children in the RCPM book and puzzle forms, combined with the finding of a strong correlation between first and second performance of the RCPM regardless of the order in which the forms were completed, shows that the alternative puzzle version is comparable to the book form in measuring reasoning ability. Past studies have reported that three factors delineate performance on the RCPM: continuous and discrete pattern completion, pattern completion through closure, and concrete abstract reasoning [23,36,37]. The high correlation between the book and puzzle forms found in the current study suggests that these constructs are maintained in the puzzle version.Study 2 demonstrated that children with severe ID who were administered the puzzle form showed a performance advantage, as compared to those who were administered the book form. The findings suggest that the puzzle form provides a better indicator of learning potential than the book form in children with severe ID. We suggest that the performance advantage observed for the puzzle form is due to its unique features designed specifically to maintain attention and increase completion rate, though have not tested this suggestion directly. This is consistent with previous studies that have shown that added motivational techniques increased performance [5,18]. However, the current study does provide evidence that attention can be engaged while maintaining the underlying constructs being measured. Thus, it is likely that the puzzle form does not demand additional cognitive processing on children with severe ID, but increases sustained attention on the task in comparison to the book form. If this were the case, it would suggest that the puzzle form effectively engages cognitive ability of children with severe ID through the integration of motor and sensory based learning but only when the child directs their own responses. This is also advantageous as it potentially useful as it puts the emphasis on the test to be able to engage children with severe ID rather than requiring the administrator efforts to promote engagement in the child during the testing. For example, a study found that certain adult style of interaction, such as following a child's line of action instead of trying to re-direct it enhances social engagement in children with ASD [14].The greater completion rate for the puzzle form than the book form for the children with severe ID in study 2 suggest that the ability to sustain attention and maintain motivation is a factor contributing to performance on cognitive ability measures for children with severe ID. This is supported by previous research showing that salience of variables engaging attention are highly correlated with measures of intelligence [38,39]. The findings challenge the clinical assumption that task incompletion reflects the inability to maintain attention and indicates limits of cognitive ability. Increasing task completion rate in children with severe ID is also important as it allows better comparison of cognitive ability to children with TD.Alternatively, the performance advantage of the puzzle form may be due to the greater completion rate for children who were administered the puzzle form, compared to the book form. It can be argued that the puzzle form produces a performance advantage because the physical placement of response pieces reduces the mental function of abstractly visualizing the chosen piece in the missing area [23]. Unlike the results of the study by Carlson and Wiedl [23], a trial and error approach was not permitted and hence this cannot be the source of increased performance when using the puzzle form. In addition, the performance advantage in the puzzle form was only demonstrated by children of the same mental age, some with ID and some developing normally, which could suggest that the puzzle form maintained attention and motivation in those with severely limited attentional resources.Given that more ID children were able to complete the puzzle form than the book form, it is possible that the performance advantage of the puzzle form was associated with an increased opportunity to select responses, as oppose to heightened task engagement. As the RCPM is a multiple choice task, the more items an individual completes, even at random, the greater the possibility of obtaining a higher overall scores. However, this is unlikely as additional analyses showed that the performance advantage of the puzzle form was maintained even when only those children who completed either RCPM form were included. However, this performance advantage was not observed in the DS and ID groups (also in the DS group when all participants were included regardless of whether they completed the RCPM or not). These non-significant findings are likely to reflect a Type II error and may be due to the small number of participants in the DS and ID groups. Future studies should examine more closely the effect of responses due to chance when completing the RCPM, specifically error-type analysis reflecting problem solving strategies in children with ID [40].Profound deficits often make the assessment of children with severe ID very difficult, and the characteristics of standardized intelligence tests do not take into consideration such deficits. The current study indicates that children with ID perform better on the puzzle form of the RCPM and suggests that it is a better indicator of problem solving ability in children with severe ID than the book form. The puzzle form has proven to give a useful measure of RCPM in children with ID as it considers the degree of intellectual disability and severity of the language deficit, as well as engage attention and motivation while limiting distractions. Hence, this study supports the use of the puzzle form in clinical and educational research settings in place of the book form, as a better measure of reasoning ability in children with severe ID and in clinical settings for monitoring treatment progress, as a component of a battery of tests.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsKB and NG created the puzzle form of the RCPM and carried out the testing of participants and collation of data and manuscript drafting. SC designed the experiment and with DC has played a role in guiding the statistical analysis and manuscript improvement. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2528155",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528155\nAUTHORS: S A McDonald, S J Hutchinson, S M Bird, C Robertson, P R Mills, J F Dillon, D J Goldberg\n\nABSTRACT:\nWe investigated trends in first-time hospital admissions and deaths attributable to hepatocellular carcinoma (HCC) in a large population-based cohort of 22 073 individuals diagnosed with hepatitis C viral (HCV) infection through laboratory testing in Scotland in 1991–2006. We identified new cases of HCC through record-linkage to the national inpatient hospital discharge database and deaths registry. A total of 172 persons diagnosed with HCV were admitted to hospital or died with first-time mention of HCC. Hepatocellular carcinoma incidence increased between 1996 and 2006 (average annual change of 6.1, 95% confidence interval (CI): 0.9–11.6%, P=0.021). The adjusted relative risk of HCC was greater for males (hazard ratio=2.7, 95% CI: 1.7–4.2), for those aged 60 years or older (hazard ratio=2.7, 95% CI: 1.9–4.1) compared with 50–59 years, and for those with a previous alcohol-related hospital admission (hazard ratio=2.5, 95% CI: 1.7–3.7). The risk of individuals diagnosed with HCV developing HCC was greatly increased compared with the general Scottish population (standardised incidence ratio=127, 95% CI: 102–156). Owing to the advancing age of the Scottish HCV-diagnosed population, the annual number of HCC cases is projected to increase, with a consequent increasing burden on the public healthcare system.\n\nBODY:\nThe aetiology of hepatocellular carcinoma (HCC) includes excessive alcohol consumption and chronic infection with the hepatitis B or C virus. The incidence of HCC has increased in developed countries worldwide (Deuffic et al, 1999; El-Serag and Mason, 1999; Law et al, 2000), but data are limited on its cause. However, modelling initiatives have projected increasing numbers of HCC deaths attributable to hepatitis B and/or hepatitis C virus (HCV) infection (Deuffic-Burban et al, 2004, 2007; Sweeting et al, 2007).In Scotland, HCV is responsible for a significant burden on healthcare (Hutchinson et al, 2006). Meeting the public health challenge of providing sufficient resources for treatment and care of patients with HCV requires up-to-date data on morbidity and mortality. However, national surveillance systems for detecting advanced HCV-related liver disease are currently lacking in the United Kingdom and elsewhere. The existence of high-quality national HCV diagnosis and hospital discharge databases provided the opportunity to use record-linkage techniques to investigate the annual number of new diagnoses of, and deaths from, HCV-related HCC.Our principal goal was to investigate trends in the annual incidence of HCC in the entire HCV-diagnosed population in Scotland through record linkage to hospital admission and deaths records. We also aimed to confirm previously reported associations between risk factors and progression to HCC. Up-to-date information regarding factors affecting the rate of development of HCC in individuals diagnosed with HCV is needed for informing clinical management and care, and for refining guidelines for early detection.Materials and methodsThe design was an observational record-linkage study involving national HCV diagnosis and hospital discharge databases and the national deaths and cancer registries, and subsequent analyses of the incidence of HCC among a retrospective cohort of all persons diagnosed with HCV infection. HCV-related HCC incidence was calculated using hospital discharge data, as this was available until 2006, and validated against cancer registrations (available until 2004).Health Protection Scotland (HPS) maintains a database of all persons who have been diagnosed as HCV positive in Scotland since testing commenced in 1991 (Shaw et al, 2003); laboratory detection of hepatitis C antibody is a requirement for inclusion. This database contains the following non-named information: surname soundex code, forename initial, date of birth, sex, and the first part of the postcode of residence, as well as data concerning risk activities. As of 31 December 2006, the database contained records for 22 073 persons diagnosed as HCV positive (HPS, 2007), who comprise the study population.The Scottish Morbidity Records (SMR01) is an episode-based patient record held by Information Services Division (ISD) of all general acute inpatient and day case hospital discharges. Discharge diagnoses use International Classification of Diseases (ICD) Ninth Revision for discharges from 1989 to 1995, and Tenth Revision for discharges from 1996 to 2006. The Scottish Morbidity Cancer Records (SMR06) is a national database containing all cancer notifications in Scotland; data include date of registration and cancer site (but not aetiology) and were available up to the end of 2004. ISD routinely combines the SMR01 and SMR06 data with death registrations held by the General Register Office for Scotland to form a linked data set.Linkage of records between the HCV diagnosis database and the previously linked SMR01/SMR06/deaths registry was carried out by ISD using probabilistic record-linkage techniques (Kendrick and Clarke, 1993). ISD had previously estimated the error rate (either false positives or false negatives) of their procedure to be less than 5% (Kendrick and Clarke, 1993). The resulting linked data set was anonymised before transfer to HPS for analysis. Linkages were approved by the Privacy Advisory Committee, which advises on confidentiality issues involving data held on NHS Scotland patients.The main outcome of interest was the annual numbers of first hospital admissions (or death, if no previous admission) with mention of HCC. Combining admission and death information to estimate the annual incidence of HCC is justified because of the relatively short lag (see Results) between first HCC admission and death. Diagnosis/cause-of-death codes indicated the development of HCC; specifically, ICD-10 C22.0 or ICD-9 155.0 mentioned as either a primary or secondary discharge diagnosis in a linked SMR01 hospitalisation record, or as either the underlying or a contributing cause of death in a linked death record. The terms admission and discharge are used interchangeably; it is assumed that discharge diagnosis codes encode the reason(s) for admission.Data analysisNo probabilistic linkages to the linked SMR01/06/death records were achieved if the HCV record lacked date of birth and two or more other identifiers. By these criteria, records for 1324 out of 22 073 persons had insufficient identifiers and thus were excluded from further analyses.We carried out two types of analyses. First, annual trends in the numbers of persons diagnosed with HCV developing HCC were estimated using Poisson regression. We analysed annual trends in counts rather than rates because the expansion of the HCV diagnosis database since its inception – with increasing proportions of persons at earlier stages of disease progression being identified and added – means that rates calculated for recent years would reflect more outcome-free person-years of follow-up than rates for earlier years. The eligible population was restricted to those with an HCV diagnosis date before or within 1 year after their first hospital admission for HCC. Data for individuals for whom first mention of HCC occurred more than 1 year before their HCV diagnosis date were excluded (n=6), as were individuals who died from any cause more than 1 year before HCV diagnosis (n=23). Of the 20 749 persons diagnosed with HCV with sufficient identifiers for record linkage, 20 720 met this 1-year constraint. Analysis of annual counts was restricted to the period 1996–2006, owing to limited HCV antibody testing before 1996. We additionally compared the annual incidence of diagnosed HCV-related HCC derived from the hospital discharge/death records linkage with the corresponding annual numbers of linked SMR06 cancer registrations over the period 1996–2004.Second, an analysis of time from HCV diagnosis to first admission/death with mention of HCC was undertaken. The observation period was defined to start 14 days after HCV diagnosis; thus records for persons diagnosed within the 14-day period before 31 December 2006 (n=44) and persons first hospitalised for HCC or who died of any cause before HCV diagnosis (n=328), or within 14 days following this date (n=154), were excluded. Of the excluded records, 28 were first diagnosed as HCV positive subsequent to their first hospital admission for HCC and 300 were first diagnosed after death. Nine individuals were first hospitalised with HCC, 3 died with mention of HCC, and 142 died of other causes within 14 days of HCV diagnosis. Confining analysis to persons who are outcome free at the start of follow-up reduces bias due to an increased risk of hospitalisation at the time of diagnosis for patients presenting with established disease. Time at risk (in person-years) was then calculated from the start of the observation period to the earliest of first hospital admission for HCC, date of death or the end of the observation period (31 December 2006). Records for 20 223 individuals diagnosed with HCV were eligible for this analysis.We used Cox proportional hazards regression analysis to assess the strength of association between risk factors (given below) and the time to first hospital admission/death with mention of HCC. Age and whether the person had a previous admission for an alcohol-related condition were treated as time-dependent covariates; sex was included as a time-independent covariate. Previous admission with mention of hepatitis B virus (HBV) infection was not included in the multifactorial analysis, as it was unassociated with the risk of developing HCC. Human immunodeficiency virus (HIV)-co-infected status was also considered as a covariate, but record linkage to a national HIV diagnosis database indicated no HCC cases to be HIV-co-infected (McDonald et al, in press). Kaplan–Meier methods were used to estimate the age-dependent probability of developing HCC, according to sex, risk activity leading to infection, and whether or not the person had a previous admission with mention of cirrhosis or an alcohol-related condition.We computed standardised incidence ratios (SIRs) for HCC in the HCV-diagnosed population by age, sex, and calendar-year standardising to expected incidence rates derived from all HCC registrations in Scotland during 1996–2004 and the national mid-year population estimates for the same period (ISD, 2008). All data analyses were carried out using R version 2.4.0 (R Foundation for Statistical Computing, 2006).For each person diagnosed with HCV, the occurrence of at least one alcohol-related hospital episode was coded as a time-dependent variable. This involved searching the linked hospital records for alcohol-related discharges occurring before the date of first mention of HCC, or, for those with no HCC admission/death, at any time before 31 December 2006. The set of alcohol-related diagnosis codes comprised alcohol use (ICD-10 Z72.1), mental and behavioural disorders due to use of alcohol (ICD-10 F10; ICD-9: 291, 303, 305), degeneration of nervous system due to alcohol (ICD-10 G31.2, G62.1, G72.1, I42.6, K29.2; ICD-9 357.5, 425.5, 535.3), toxic effects of alcohol (ICD-10 T51.0, T51.9; ICD-9 980.0), alcoholic liver disease (ICD-10 K70.1-3; ICD-9 571.0–571.2), alcohol-induced chronic pancreatitis (ICD-10 K86.0), evidence of alcohol involvement (ICD-10 Y90-1), finding of alcohol in blood (ICD-10 R78.0; ICD-9 790.3), alcohol rehabilitation (ICD-10 Z50.2), personal history of psychoactive substance abuse (ICD-10 Z86.4; captures non-current mental/behavioural disorders due to use of alcohol), and accidental or intentional self-poisoning by and exposure to alcohol (ICD-10 X45, X65; ICD-9 E860.0, E860.9).Additional risk factors identified by previous research as predictors of the development of HCC were coded for each person. These included age (time-dependent: <50, 50–59 and 60+ years) and reported risk activity leading to infection (injecting drug use (IDU), non-IDU, not known). The non-IDU group included persons who had reported potential acquisition of HCV infection through receipt of blood or blood products, tattoo/body piercing, needlestick injury, sexual contact, or perinatal transmission. Previous hospitalisation for cirrhosis was also assessed by searching the database linkage for mention of non-biliary cirrhosis among the discharges occurring before first mention of HCC, or at any time before 31 December 2006 for those with no admission/death for HCC. Cirrhosis codes comprised alcoholic cirrhosis of liver (ICD-10 K70.3; ICD-9 571.2), cirrhosis of liver without mention of alcohol (ICD-9 571.5), and other and unspecified cirrhosis of liver (ICD-10 K74.6). The occurrence of a previous hospital discharge with mention of HBV (ICD-10 B16, B18.0, B18.1; ICD-9 070.2, 070.3) was similarly noted.ResultsThe majority (68%) of the study population were male (Table 1); at the time of HCV diagnosis, 42% were under the age of 30 years and 13% were 45 years or older (median=32 years). A total of 31% had a previous hospital discharge related to alcohol, 3.5% for cirrhosis, and 5.6% with mention of hepatitis B.Over a median follow-up of 5.6 years (range=0–22.0) for all 20 720 persons diagnosed with HCV, 172 were either admitted with first-time mention of HCC (n=145) or died without being previously hospitalised with HCC (n=27); that is, their first and only indication of HCC was recorded on the death record. The median time from HCV diagnosis date to first-time mention of HCC was 2.3 years (0.2, 1.7, and 3.5 years, for HCC calendar periods <1996, 1996–2000, and >2000, respectively). Of the 145 individuals first hospitalised with HCC, 115 subsequently died, of whom 95 had their underlying or a contributing cause of death recorded as HCC; 55% (11 out of 20) of the other deaths specified a liver-related underlying cause. Twenty-two out of 172 individuals were diagnosed with HCV following their first HCC admission/death; the majority of these diagnoses were made within 2 weeks (n=19) of first HCC admission/death.For those individuals who died with an underlying cause of HCC (tumour) subsequent to their first hospitalisation for HCC (n=83), the median lag between first-time admission and death was 81 days. Forty-four per cent (76 out of 172) of HCC cases had a previous admission for cirrhosis, and 31% (53 out of 172) had a previous alcohol-related hospitalisation. The median lag between first admission for cirrhosis and first-time mention of HCC was 2.3 years. Among only those persons hospitalised for cirrhosis, the one- and two-year cumulative risks of developing HCC following first admission for cirrhosis were 3.5 and 6%, respectively.Hepatitis C virus was listed as a discharge diagnosis in 39% (56 out of 145) of HCC admissions, and as the underlying or a contributing cause in 41% (11 out of 27) of deaths with first-time mention of HCC.Table 2 shows the number of first-time hospital admissions/deaths per year over the period 1996–2006, stratified by age group at admission/death. There was a significant increasing trend in the overall number of first-time admissions/deaths (average annual change=6.1%, 95% CI: 0.9–11.6%, P=0.021). Incorporation of a year by age-group interaction term indicated a significantly greater mean annual change in the 50–59 years age group (16.3%, 95% CI: 6.1–27.5%, P=0.028) compared with the <50 years age group.The incidence of HCC in the HCV-diagnosed population as determined from hospital discharge/death records was compared with the linked (HCV-related) SMR06 cancer registrations over the period 1996–2004. There was good agreement between the annual numbers of first-time HCC admissions/deaths and the SMR06 data. In the study period, there were 108 new entries for HCC in the cancer registry (12, 16, 4, 11, 6, 13, 15, 14, and 17 registrations for the years 1996 through 2004, respectively), 7 fewer than the 115 new cases of HCC determined from the hospital admission/death record linkage in the same period. Three of the cases absent from the linked cancer registrations were deaths without previous admission for HCC. Five of the 108 cancer registrations were not found in the hospital admission/death record linkage.Figure 1 shows the age-dependent proportion of persons with a first-time hospitalisation/death with mention of HCC as a function of sex and whether or not the person had a previous alcohol- or cirrhosis-related admission. There was no difference in the unadjusted cumulative probability of developing HCC according to previous alcohol-related admission status (log-rank test, P=0.41). In contrast, a previous admission for cirrhosis was associated with a much greater proportion of persons developing HCC (P<0.001).Table 3 shows adjusted hazard ratios derived from multifactorial Cox proportional hazards regression analysis, with sex, current age (<50, 50–50, and 60+ years), and previous alcohol-related admission as covariates. Violation of the proportional hazards assumption was assessed graphically and tested using Schoenfield residuals (global P=0.63). Males were more likely than females to develop HCC (hazard ratio=2.7, 95% CI: 1.7–4.2). Compared with the reference age group (50–59 years), older age (60+ years) was associated with an increased risk of first-time admission/death (HR=2.7, 1.9–4.1) and younger age (<50 years) with a decreased risk (HR=0.03, 95% CI: 0.02–0.05); there was a 2.5-fold increased risk associated with a previous alcohol-related admission (HR=2.5, 95% CI: 1.7–3.7).Over the period 1996–2004, a total of 1304 HCC cases were registered in Scotland (ISD, 2008). The estimated prevalence of diagnosed HCV infection among all HCC cases was therefore 8.8% (115 out of 1304; 1996–1999 only: 8.4% (45 out of 535); 2000–2004: 9.1% (70 out of 769)). The SIR for HCC was 127 (95% CI: 102–156). We also calculated separate SIRs for males and females: 118 (95% CI: 93–149) and 179 (95% CI: 106–283), respectively.Over the entire observation period (1991–2006), 108 HCV-diagnosed persons died with an underlying cause of death recorded as HCC, representing 4% of all deaths (n=2622) that occurred during this period. One- and two-year probabilities of death with mention of HCC subsequent to first-time hospitalisation for HCC were 53 and 58%, respectively; the respective one- and two-year probabilities of death from any cause were 62 and 70%. Of those who died of HCC, 39% (42 out of 108) had a previous admission for cirrhosis, and 32% (35 out of 108) had a previous alcohol-related admission.DiscussionIn this study, we generated up-to-date estimates of the annual incidence of HCC in the Scottish HCV-diagnosed population and assessed the relative risk of developing HCC associated with various variables. A key strength of this study is the use of national data sources, which provided over 120 000 person-years of follow-up.The incidence of HCC increased over the period 1996–2006. Consistent with this trend, there was an increasing trend in the number of HCV-related deaths from liver cancer in Scotland over the period 1996–2005 (Palmateer et al, 2007). Similar rising trends in the incidence of HCV-related HCC have been observed over overlapping periods in England (1996–2004; Sweeting et al, 2007) and Australia (1990–2002; Amin et al, 2007).Of those persons who died subsequent to their first hospitalisation with HCC, only 83% (95 out of 115) had HCC mentioned in their death record, suggesting that studies that rely on death registry data may underestimate HCC incidence. This is particularly relevant for modelling studies using back-calculation methods to estimate the past incidence of chronic HCV infection (Deuffic-Burban et al, 2004, 2007; Sweeting et al, 2007). Our finding that only 40% of hospitalisations and 38% of deaths with first-time mention of HCC had an HCV code listed also highlights the limitations of registry methods.In the general Scottish population, the overall incidence rate for HCC in the period 1996–2004 was 2.9 per 100 000 persons (ISD, 2008). Incidence was many times greater in the HCV-diagnosed population; over the period 1996–2004, we obtained an SIR of 127, five times larger than the SIR of 27 (95% CI: 23–31) in a New South Wales (NSW), Australia, study (Amin et al, 2006; revised SIR from J Amin, personal communication). This large discrepancy in SIRs is attributable to differences in the definition of time at risk; in the NSW study, the observation period is defined to start 1 year after HCV diagnosis, as opposed to 14 days in this study. Notably, the estimated prevalence of diagnosed HCV among all HCC cases (8.8%) in Scotland was lower than the value (13.7%) reported for NSW (Amin et al, 2007).A shift in the incidence of HCC in one age group (40–60 years) in a US population-based study has been reported (El-Serag and Mason, 1999), reflecting the delay between disease onset and HCV infection (thought to occur in the 1960s and 1970s). We observed the largest average annual change in HCC incidence (16.3%) in the 50–59 years group; for these persons, the mean age at diagnosis was 52, suggesting that infection occurred 20–25 years earlier, in the 1970s.Time-to-event analysis indicated that male sex, older age (50+ years), and the occurrence of a previous hospital discharge for an alcohol-related condition were all factors that greatly increased the relative risk of developing HCC. These results concur with previous research (Niederau et al, 1998; Schafer and Sorrell, 1999; Kuper et al, 2001).Cirrhosis is an important risk factor for HCC, with HCC developing in cirrhotic livers (irrespective of viral hepatitis infection) with an annual incidence of 3% (Colombo et al, 1991; Velazquez et al, 2003). An estimated 5–15% of chronic HCV-infected individuals develop cirrhosis within 20 years after diagnosis (Freeman et al, 2001; Seeff, 2002; Benvegnù et al, 2004). In HCV-infected patients with established cirrhosis, the annual incidence and 5-year probability of HCC developing have been estimated at 3.5% (Hutchinson et al, 2005) and 28–30% (Benvegnù et al, 2004; Sola et al, 2006), respectively.A previous admission with mention of cirrhosis was recorded for 45% of HCC cases and was followed by hospital admission/death with first-time mention of HCC at a median lag of 2.3 years. Given the relatively young age distribution of the Scottish HCV-diagnosed population (median age at diagnosis is 32) and the relatively short observation time, the number of persons developing cirrhosis will be expected to increase as this population ages. We expect to see a corresponding increase in the number of individuals diagnosed with HCV infection who progress to HCC.Previous hospital admission for an alcohol-related condition was associated with a 2.6-fold increased risk of developing HCC. Problem alcohol use, to the extent that it is captured by our proxy variable, appear to be highly prevalent in our study population; 31% of the cohort had been previously admitted to hospital with an alcohol-related diagnosis.High levels of alcohol consumption, particularly above 350 g per week, have been associated with accelerated progression to cirrhosis in chronic HCV-infected persons (Ostapowicz et al, 1998; Freeman et al, 2003; Monto et al, 2004). There is less evidence that alcohol increases the risk of developing HCC once cirrhosis is established (Adami et al, 1992; Teli et al, 1995; Velazquez et al, 2003; Planas et al, 2004; also see Miyakawa et al, 1996; Tsutsumi et al, 1996), suggesting an indirect mechanism by which alcohol increases the risk of HCC (Kuper et al, 2001). Given the strong association between excessive alcohol intake and cirrhosis, one measure for reducing the risk of HCC in the HCV-infected population is to limit alcohol intake.Development of HCC in HCV-infected persons usually requires 15–25 years (Schafer and Sorrell, 1999; Freeman et al, 2001); the median lag between HCV diagnosis and hospitalisation/death with first mention of HCC in our study was much shorter, 2.3 years. Our study population, in particular those aged 40 and over at diagnosis, were likely tested and diagnosed as HCV antibody positive many years after infection, and in numerous cases, diagnosis was concurrent to or shortly followed first hospital admission with HCC. Given accurate data on the date of infection, we would expect the time to HCC development to be more consistent with previous studies.The incidence of HCV-related HCC may have been underestimated due to (i) insufficient identifiers in the HCV Diagnosis database for record linkage and (ii) an unknown, but likely small, number of HCC cases that were not tested (and so never diagnosed) for HCV infection during 1996–2006.In conclusion, the significance of excess risk of HCC and the trend for increasing annual numbers of 50- to 59-year-olds developing HCC are consistent with ageing of the members of the Scottish HCV-diagnosed population and the consequent progression to severe liver disease. Our study has highlighted the growing burden on public healthcare resources from HCV-related outcomes such as HCC.\n\nREFERENCES:\n1. Adami HO, Hsing AW, McLaughlin JK, Trichopoulos D, Hacker D, Ekbom A, Persson I (1992) Alcoholism and liver cirrhosis in the etiology of primary liver cancer. Int J Cancer\n51: 898–9021639537\n2. Amin J, Dore GJ, O'Connell DL, Bartlett M, Tracey E, Kaldor JM, Law MG (2006) Cancer incidence in people with hepatitis B or C infection: a large community-based linkage study. J Hepatol\n45: 197–20316684579\n3. Amin J, O'Connell D, Bartlett M, Tracey E, Kaldor J, Law M, Dore G (2007) Liver cancer and hepatitis B and C in New South Wales, 1990–2002: a linkage study. Aust NH Z J Public Health\n31: 475–482\n4. Benvegnù L, Gios M, Boccato S, Alberti A (2004) Natural history of compensated viral cirrhosis: a prospective study on the incidence and hierarchy of major complications. Gut\n53: 744–74915082595\n5. Colombo M, de Franchis R, Del Ninno E, Sangiovanni A, De Fazio C, Tommasini M, Donato MF, Piva A, Di Carlo V, Dioguari N (1991) Hepatocellular carcinoma in Italian patients with cirrhosis. N Engl J Med\n325: 675–6801651452\n6. Deuffic S, Buffat L, Poynard T, Valleron AJ (1999) Modeling the hepatitis C virus epidemic in France. Hepatology\n29: 1596–160110216148\n7. Deuffic-Burban S, Wong JB, Valleron AJ, Costagliola D, Delfraissy JF, Poynard T (2004) Comparing the public health burden of chronic hepatitis C and HIV infection in France. J Hepatol\n40: 319–32614739105\n8. Deuffic-Burban SF, Poynard TF, Sulkowski MS, Wong JB (2007) Estimating the future health burden of chronic hepatitis C and human immunodeficiency virus infections in the United States. J Viral Hepat\n14(2): 107–11517244250\n9. El-Serag HB, Mason AC (1999) Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med\n340: 745–75010072408\n10. Freeman AJ, Dore GJ, Law MG, Thorpe M, Von Overbeck J, Lloyd AR, Marinos G, Kaldor JM (2001) Estimating progression to cirrhosis in chronic hepatitis C virus infection. Hepatology\n34: 809–81611584380\n11. Freeman AJ, Law ME, Kaldor JM, Dore GJ (2003) Predicting progression to cirrhosis in chronic hepatitis C virus infection. J Viral Hepat\n10: 285–29312823595\n12. Health Protection Scotland (HPS) (2007) Surveillance of known hepatitis C antibody positive cases in Scotland: results to 31 December 2006. HPS Weekly Report\n41: 181–186\n13. Hutchinson SJ, Bird SM, Goldberg DJ (2005) Modeling the current and future disease burden of hepatitis C among injection drug users in Scotland. Hepatology\n42: 711–72316116637\n14. Hutchinson SJ, Roy KM, Wadd S, Bird SM, Taylor A, Anderson E, Shaw L, Codere G, Goldberg DJ (2006) Hepatitis C virus infection in Scotland: epidemiological review and public health challenges. Scott Med J\n51: 8–15\n15. Information Services Division (ISD) (2008) Liver cancer. http://www.isd.scot.nhs.uk/isd/1478.html (Specific data on hepatocellular carcinoma incidence extracted April 2008)\n16. Kendrick S, Clarke J (1993) The Scottish record linkage system. Health Bull (Edinb)\n51: 72–798514493\n17. Kuper H, Ye W, Broomé U, Romelsjö A, Mucci LA, Ekbom A, Adami H-O, Trichopoulos D, Nyrén O (2001) The risk of liver and bile duct cancer in patients with chronic viral hepatitis, alcoholism, or cirrhosis. Hepatology\n34: 714–71811584367\n18. Law MG, Roberts SK, Dore GJ, Kaldor JM (2000) Primary hepatocellular carcinoma in Australia, 1978–1997: increasing incidence and mortality. Med J Aust\n173: 403–40511090031\n19. McDonald SA, Hutchinson SJ, Bird SM, Mills PR, Dillon J, Bloor M, Robertson C, Donaghy M, Hayes P, Graham L, Goldberg DJ (in press) A population-based record-linkage study of mortality in hepatitis C diagnosed persons with and without HIV coinfection in Scotland. Stat Methods Med Res\n20. Miyakawa H, Izumi N, Marumo F, Sato C (1996) Roles of alcohol, hepatitis virus infection, and gender in the development of hepatocellular carcinoma. Alcohol Clin Exp Res\n20: 91a–94a\n21. Monto A, Patel K, Bostrum A, Pianko S, Pockros P, McHutchison JG, Wright TL (2004) Risks of a range of alcohol intake on hepatitis C-related fibrosis. Hepatology\n39: 826–83414999703\n22. Niederau C, Lange S, Heintges T, Erhardt A, Buschikamp B (1998) Prognosis of chronic hepatitis C: results of a large, prospective cohort study. Hepatology\n28: 1687–16959828236\n23. Ostapowicz G, Watson KJ, Locarnini SA, Desmond PV (1998) Role of alcohol in the progression of liver disease caused by hepatitis C virus infection. Hepatology\n27: 1730–17359620350\n24. Palmateer NE, Hutchinson SJ, McLeod A, Codere G, Goldberg DJ (2007) Comparisons of deaths related to hepatitis C and AIDS in Scotland. J Viral Hepat\n14: 870–87418070290\n25. Planas R, Balleste B, Alvarez MA, Rivera M, Montoliu S, Galeras JA, Santos J, Coll S, Morillas RM, Sola R (2004) Natural history of decompensated hepatitis C virus-related cirrhosis. A study of 200 patients. J Hepatol\n40: 823–83015094231\n26. R Development Core Team (2006) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org\n27. Schafer DF, Sorrell MF (1999) Hepatocellular carcinoma. Lancet\n353: 1253–125710217098\n28. Seeff LN (2002) Natural history of chronic hepatitis C. Hepatology\n36: S35–S4612407575\n29. Shaw L, Taylor A, Roy KM, Cameron SO, Burns S, Molyneaux P, McIntyre P, Codere G, Goldberg D (2003) Establishment of a database of diagnosed HCV-infected persons in Scotland. Commun Dis Public Health\n6: 305–31015067856\n30. Sola R, Alvarez MA, Balleste B, Montoliu S, Rivera M, Miquel M, Cirera I, Morillas RM, Coll S, Planas R (2006) Probability of liver cancer and survival in HCV-related or alcoholic-decompensated cirrhosis. A study of 377 patients. Liver Int\n26: 62–7216420511\n31. Sweeting MJ, De Angelis D, Brant LJ, Harris HE, Mann AG, Ramsay ME (2007) The burden of hepatitis C in England. J Viral Hepat\n14: 570–57617650291\n32. Teli MR, Day CP, Burt AD, Bennet MK, James OF (1995) Determinants of progression to cirrhosis or fibrosis in pure alcoholic fatty liver. Lancet\n346: 987–9907475591\n33. Tsutsumi M, Ishizaki M, Takada A (1996) Relative risk for the development of hepatocellular carcinoma in alcoholic patients with cirrhosis: a multiple logistic-regression coefficient analysis. Alcohol Clin Exp Res\n20: 758–7628800396\n34. Velazquez RF, Rodriguez M, Navascues CA, Linares A, Perez R, Sotorrios NG, Martinez I, Rodrigo L (2003) Prospective analysis of risk factors for hepatocellular carcinoma in patients with liver cirrhosis. Hepatology\n37: 520–52712601348"
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+    "id": "PMC2528256",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528256\nAUTHORS: Cynthia Lee Richard, Jonathan Blay\n\nABSTRACT:\nChemokines are peptide mediators involved in normal development,\nhematopoietic and immune regulation, wound healing, and\ninflammation. Among the chemokines is CXCL12, which binds\nprincipally to its receptor CXCR4 and regulates leukocyte\nprecursor homing to bone marrow and other sites. This role of\nCXCL12/CXCR4 is “commandeered” by cancer cells to facilitate the\nspread of CXCR4-bearing tumor cells to tissues with high CXCL12\nconcentrations. High CXCR4 expression by cancer cells predisposes\nto aggressive spread and metastasis and ultimately to poor patient\noutcomes. As well as being useful as a marker for disease\nprogression, CXCR4 is a potential target for anticancer therapies.\nIt is possible to interfere directly with the CXCL12:CXCR4 axis\nusing peptide or small-molecular-weight antagonists. A further\nopportunity is offered by promoting strategies that downregulate\nCXCR4 pathways: CXCR4 expression in the tumor microenvironment is\nmodulated by factors such as hypoxia, nucleosides, and\neicosanoids. Another promising approach is through targeting PPAR\nto suppress CXCR4 expression. Endogenous PPARγ such as 15-deoxy-Δ12,14-PGJ2 and synthetic agonists such as the\nthiazolidinediones both cause downregulation of CXCR4 mRNA and\nreceptor. Adjuvant therapy using PPARγ agonists may, by\nstimulating PPARγ-dependent downregulation of CXCR4 on cancer cells, slow the rate of metastasis and impact beneficially on\ndisease progression.\n\nBODY:\n1. INTRODUCTIONThe regulation of the distribution of motile\ncells in both normal and disease situations depends upon a variety of peptide\nand nonpeptide mediators, which stimulate cell movement by both directed\n(chemotaxis) and nondirected (chemokinesis) mechanisms. Amongst these mediators\nare the chemokines, a class of peptide mediators that play critical roles in\nnormal development, regulation of the hematopoietic and immune systems in the\nadult, and in repair processes such as wound healing and inflammation. Among\nthe different chemokines is the stromal cell-derived factor-1 (SDF-1), which is\nnow known as CXCL12. CXCL12 binds principally to the receptor CXCR4, although\nit also acts through the more-recently-described receptor CXCR7 [1]. This\nreview describes the roles of CXCL12 and CXCR4 in normal tissue functions and\nin cancer, and suggests that the regulation of CXCR4 expression by PPARγ may\nemerge to be a unique avenue by which a key receptor involved in cancer cell\nmetastasis can be suppressed in a way that will assist with disease therapy.2. CHEMOKINES AND THEIR RECEPTORS IN\nCELL REGULATIONChemokines\nare low-molecular-weight peptide ligands involved in the trafficking of\nleukocytes and other motile cells [2, 3]. \nThere are four major groups of chemokines, the CXC, CC, C and CX3C\nchemokines, categorized as such on the basis of their number and spacing of\nconserved cysteine residues [2, 4]. The nomenclature\nof chemokines (e.g., “CXCL12\") is made up of their subclass (CXC, CC, etc.)\nfollowed by “L” for ligand, and a specific number [2, 3].The receptors for\nchemokines are cell-surface, seven-transmembrane G protein-coupled receptors [2].\nThe naming of these receptors (e.g., “CXCR4\") is based on the subclass of\nchemokine that the receptor recognizes, followed by “R” for receptor and a\nnumber (which need not correspond to the number assigned to its cognate ligand(s)). There are 19 well-recognized chemokine\nreceptors (e.g., CXCR1-6, CCR1-10, CX3CR1, and XCR1) [1, 5]. Many\nchemokine receptors have more than one known ligand, and many chemokines can\nactivate more than one receptor. Thus,\nthere is much promiscuity in chemokine/receptor signaling.Chemokines bind within\nthe extracellular domain of the chemokine receptor, which comprises the\nN-terminus and three extracellular loops [3]. \nThe intracellular domain, which consists of three loops and the\nC-terminus, associates with G proteins that, upon activation, lead to\ninhibition of adenylyl cyclase activity [3]. Typical cellular consequences of\nchemokine binding include changes in gene expression, cell polarization, and\nchemotaxis (directed cell migration) [4].Chemokines play a\nmajor role in regulating the migration of cells of the immune system, leading\nto the modulation of immune responses. \nTheir exact role depends on the expression pattern of receptors on\nspecific leukocyte subsets [2] but encompasses the regulation of lymphocyte\ntrafficking, lymphoid tissue development, Th1/Th2 modulation, and the effecting\nof inflammatory reactions. Chemokine receptors are also found on other cell\ntypes, and play a part in stem cell recruitment and angiogenesis, in\ndevelopment and wound healing [4]. When such pathways are subverted in\nneoplastic cells, chemokines take over prominent roles in the metastatic\nprocess, both in terms of the dissemination of cells from primary tumors and in\ngrowth of the cancer at metastatic sites. As we will see, this is the case for\nCXCR4.3. THE CHEMOKINE RECEPTOR CXCR4 AND\nITS LIGAND CXCL12 (SDF-1)The receptor now\nknown as CXCR4 was cloned in 1994, and was originally given the name leukocyte-expressed\nseven-transmembrane domain receptor (LESTR) due to its abundant expression in\nseveral leukocyte populations [6]. It\nwas independently cloned by others and named “fusin” because of its ability to\nact as a coreceptor for HIV fusion and entry [7]. It further has the designation “CD184” as\npart of the cluster of differentiation antigens found on activated leukocytes. LESTR/fusin/CD184\nwas originally considered to be an orphan receptor. However, the chemokine CXCL12, originally termed\nstromal cell-derived factor 1 (SDF-1), was shown by two independent research\ngroups to be a ligand for LESTR/fusin/CD184, and the name CXCR4 was proposed [8, 9]. The CXCR4 gene is constitutively expressed, and CXCR4 protein has been\ndetected on many leukocytes, including lymphocytes, monocytes, NK cells, and\ndendritic cells; as well as on vascular smooth muscle cells, endothelial cells,\ncells lining the gastrointestinal tract, microglia, neurons, and astrocytes [10–13].\nUntil recently, CXCR4 was considered to be the only receptor for CXCL12, but\nthe previous orphan receptor RDC1 is now recognized as an additional CXCL12 receptor,\nfor which the name CXCR7 has been given [1]. \nCXCL12 itself is widely expressed at different levels in many tissues [14].4. CXCL12 AND CXCR4 IN NORMAL TISSUE FUNCTIONThe interplay between CXCL12 and CXCR4 is\ncritical to normal development. Indeed (and unlike mice deficient in other\nchemokine/receptors) mice lacking CXCL12 or CXCR4 die in utero or shortly after birth [2, 15–17]. CXCL12/CXCR4 signaling is required during the\ndevelopment of the hematopoietic, cardiac, vascular, and nervous systems. Absence\nof this axis in embryonic life leads to defects in bone marrow myeloid cell\nformation, cardiac dysfunction due to impaired ventricular septum formation,\nand developmental defects in the cerebellum and in the vasculature of the\ngastrointestinal tract [15–17].In the normal\nadult, CXCL12 and CXCR4 are involved in the homing and retention of\nhematopoietic progenitor cells in the bone marrow. These progenitor cells express high levels of\nCXCR4, and are attracted to CXCL12 produced by stromal cells in specialized bone\nmarrow niches [18]. Activating mutations\nof the CXCR4 gene lead to aberrant\nretention of myeloid cells within the bone marrow [19]. CXCL12 also acts as a\nchemoattractant for stem cells and some differentiated cells in the pathological\ncontexts of inflammation and tissue regeneration/repair [20–24]. It is this\nfunction of controlling cell migration and homing that is subverted in cancer.5. CXCL12 AND CXCR4 IN CANCER\nMETASTASIS AND GROWTHIn many ways, the\nprocess of metastasis is similar to leukocyte and stem cell trafficking,\nprocesses which involve the CXCL12/CXCR4 axis [20]. Cancer cells that express CXCR4 exploit the\nsame signaling pathway, leading to homing and retention in tissues that are\nrich in CXCL12.The foundation for\nour appreciation of the role that CXCR4 and CXCL12 may play in cancer\nmetastasis was set in 2001, when a landmark study by Albert Zlotnik's\ngroup demonstrated the importance of the CXCL12/CXCR4 axis in site-specific\nmetastasis of breast cancer [25]. In that study, it was found that CXCR4\nexpression was low or undetectable in normal epithelial cells, but consistently\nupregulated in breast cancer cell lines and primary breast cancer cells at both\nthe mRNA and protein level. Human breast\ncarcinoma cells that expressed high levels of CXCR4 underwent morphological\nchanges and migrated directionally in response to CXCL12, indicating that the\nCXCR4 receptor was active. Crucially,\nthe ligand CXCL12 was highly expressed in tissues taken from human organ sites\nto which breast cancer cells metastasize, including lymph nodes, lung, liver,\nand bone marrow, but expressed at low levels in tissues that represent rare\nsites of metastasis, including the kidney, skin, and muscle. The ability of MDA-MB-231 human breast cancer\ncells (a cell line that is metastastic in experimental models) to migrate\ntowards protein extracts of lung and liver, or to produce lung and lymph node\nmetastasis after tail-vein injection or orthotopic implantation, was inhibited by\nneutralizing anti-CXCR4 and/or anti-CXCL12 antibodies. These findings were the first to show the\nbiological importance of this chemokine/receptor pair in the evolution and\nspread of cancer.Since that time,\nthe CXCL12/CXCR4 axis has been shown to be important in the progression and\nspread of more than 25 different cancers. Our present knowledge is based on\n(i) studies in cellular and animal experimental models, (ii) surveys of human tissues at\ndifferent stages of cancer progression, and (iii) population-based studies of morbidity\nand survival. A summary of present data is shown in Table 1.CXCR4 has been\nshown to be expressed at high levels on cells of all of the major adult solid\nepithelial cancers (breast, colorectal, lung, ovary, prostate, etc.). The\nability of the cells to colonize other tissues by gaining advantage from\nCXCR4-dependent mechanisms depends on the presence of CXCL12 in the tissue\nfluid. Various studies have shown significant CXCL12 concentrations in the\nfluid-filled cavities through which many cancers disseminate, and at tissue\nlocations in which metastases characteristically develop. Biologically,\nsignificant CXCL12 levels have been\nfound in peritoneal ascites from ovarian cancer patients [26], pleural\neffusions in lung cancer [27], lymph nodes, bone, and lungs as well as other\ntissues [25, 28, 29].Detailed studies\nof the cellular interactions involved in the metastasis of prostate cancer\ncells to bone [29] have shown that the interaction of CXCL12 with CXCR4 plays a\nmajor role in successive steps in the metastatic process. Human osteoblasts\nexpress CXCL12 mRNA and protein, whereas prostate cancer cells express CXCR4\nmRNA and receptor. Prostate cancer cells\nthat have become disseminated into the circulation respond to the CXCL12-CXCR4\npathway by enhanced adherence to the bone marrow endothelium and migration\nacross endothelial barriers and basement membranes, ultimately adhering to\ncomponents of the bone marrow in response to a CXCL12 gradient [29]. CXCL12\nfrom osteoblasts has also been shown to act on CXCR4 to induce release of IL-6\nfrom human squamous cell carcinoma cells to promote osteoclastogenesis [30].As well as\npromoting the migration of cancer cells and their invasion through physical\nbarriers as well as adherence to target structures, CXCL12 can act upon CXCR4\non the cancer cells to promote cancer cell growth along with other mitogenic\nfactors. This has been shown in cells from colorectal [31], prostate [32], and\novarian [33] cancers. Furthermore, CXCL12 can promote cancer\ndissemination indirectly by enhancing the vascular supply, since the\nCXCL12/CXCR4 axis may also promote tumor angiogenesis. Vascular endothelial growth factor (VEGF) and\nCXCL12 have been shown to increase angiogenesis synergistically in an in vivo Matrigel assay and to promote\nproliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro [34].6. THE EFFECT OF CXCL12 ON CELLULAR PROCESSESActivation\nof CXCR4 produces specific cellular changes that are consistent with a\nmigratory and invasive cell phenotype. Exposure of cells to CXCL12 produces upregulation\nof matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9 [25, 26, 29, 35–39].\nIn addition, CXCL12 enhances adhesion to components of the extracellular matrix\nsuch as fibronectin, laminin, and collagen types I/III [37, 40], or to other\ncell types (e.g., endothelial or bone marrow stromal cells) [29, 41, 42]. These\nchanges are mediated in large part by integrin signaling [29, 43, 44]. Many\nsignaling pathways are activated by CXCL12 downstream of CXCR4 in cancer\ncells. For example, CXCL12 has been\nshown to increase ERK1/2 phosphorylation [30, 31, 49, 0.70, 76.78, 79], Akt\nphosphorylation [50, 77.88], and PI3K activation [45].7. CXCR4 IN BREAST CANCERCXCR4 is expressed\nat a low level in normal breast epithelium but becomes more strongly expressed\nin the early stages of carcinogenesis, showing both a more intense immunohistochemical\nstaining pattern and an altered cellular localization in studies of human\nductal carcinoma in situ (DCIS)\n[46, 47]. An extensive tissue microarray study of 1808 invasive breast\ncarcinomas and 214 pre-invasive breast samples linked to clinical data has\nshown that the level of CXCR4 expression can be linked to tumor progressivity\n(tumor grade and lymph node status) and to other prognostic factors such as\nHER2 expression and hormone receptor (ER and PR) negativity, as well as to\npatient survival [46]. These observations in human tissues have led to the view\nthat CXCR4 provides a selective advantage to newly formed neoplastic cells in\nthe early primary breast tumor as well as being important to later invasion and\nmetastasis [13, 46–48]. This is consistent with observations in mouse models of\nbreast cancer in which interventions affecting CXCR4 reduced both growth of the\nprimary tumor and metastasis [49].Prominent CXCR4\nexpression is a feature of all major histological forms of invasive breast\ncancer, including ductal, lobular, mucinous [46], and the distinctive and\nhighly aggressive inflammatory form of the disease [50]. Several independent\nstudies have shown that the extent and pattern of CXCR4 expression is related\nto axillary lymph node involvement in different forms and stages of breast\ncancer [28, 51–53]. CXCR4 positivity has also been noted as a key feature of\nbreast carcinoma metastasis to bone [54] and brain [55]. The power of CXCR4 as\na marker for lymph node metastasis can be greatly increased by concurrently\nexamining the expression of additional markers such as VEGF, MMP-9, and CCR7 [38, 56].\nFurthermore, CXCR4 is also one of a subset of markers (the others are uPAR,\nS100A4, and vimentin) that define highly aggressive and invasive breast\ncarcinoma cells that are associated with malignant pleural or peritoneal\neffusions in breast cancer patients [57]. CXCR4 expression is therefore a general\nmarker for the spread of breast cancer to its secondary sites, and for aggressive\nstages of the disease.There is evidence\nnot only for the use of CXCR4 as a general marker for the progression and\nmetastasis of breast cancer, but also for the identification of individual tumor\ncells as they are homing from the primary tumor to secondary sites as patients\ndevelop metastatic disease. Individual CXCR4-expressing tumor cells have been\nfound in the peripheral blood of breast cancer patients [102], and CXCR4\nexpression in breast cancer has been associated with the presence of individual\ntumor cells in the bone marrow of patients [103].8. CXCR4 IN COLORECTAL CANCERCXCR4 is\nabundantly expressed by colorectal carcinoma cells [104, 105]. The involvement of CXCR4 expression in\ncolorectal cancer progression was first shown by Roos and colleagues [71]. CT-26 mouse colon carcinoma cells were\ntransfected with CXCL12 extended with a Lys-Asp-Glu-Leu (KDEL) sequence. The KDEL receptor functions to retain\nresident endoplasmic reticulum (ER) proteins, which contain a C-terminal KDEL\nsequence, in the ER. With this\n“intrakine approach,\" CXCL12-KDEL binds to the KDEL receptor and is retained in\nthe ER, and CXCR4 protein which binds to CXCL12 is also retained in the ER,\npreventing its expression at the cell-surface [71, 106]. This approach was first developed as a\nstrategy to reduce HIV infection [107]. After intrasplenic injection, CXCL12-KDEL-transfected CT-26 cells, which had reduced cell-surface CXCR4\nprotein expression, did not form liver metastases, whereas control cells did [71]. The incidence of lung metastasis was also\nreduced with CXCL12-KDEL-transfected cells, and survival was increased. Interestingly, unlike Zlotnik's group, who\nhad suggested that CXCR4 expression was necessary for the movement of tumor cells\nto secondary sites [25], Zeelenberg and colleagues found that CXCR4 expression\nwas not required for migration of CT-26 colorectal tumor cells to the lungs,\nbut rather for tumor expansion at secondary sites [71]. Therefore, these authors concluded that CXCR4\nis necessary for the outgrowth of colon cancer micrometastases.Ottaiano\nand colleagues found that CXCR4 was overexpressed in human colorectal carcinoma\ntissues compared to normal tissues [40]. \nCell-surface CXCR4 protein was also expressed at high levels on SW620,\nSW48, and SW480 colorectal carcinoma cells, and at moderate levels on Caco-2\nand LoVo cells. CXCL12 enhanced the\nchemotaxis of SW480 cells as well as their adhesion to fibronectin and collagen\ntype I/III, and both effects were blocked with an anti-CXCR4 neutralizing\nantibody. CXCL12 also induced\ncytoskeletal changes, proliferation, and ERK1/2 phosphorylation in SW480 cells. Similarly, Schimanski and colleagues found that\nSW480, SW620, and HT-29 colorectal carcinoma cells expressed CXCR4 protein, as\ndid colorectal carcinoma tissue samples [72]. \nCXCL12 induced the chemotaxis of SW480 and SW620 cells. Kim and colleagues found that in patients with colorectal cancer with liver\nmetastases, higher CXCR4 expression was found on metastatic tissues compared to\nthe primary tumor [73]. Furthermore,\nelevated CXCR4 expression in colorectal cancer is associated with disease\nprogression and reduced survival [40, 72, 73, 75].9. THE UTILITY OF CXCR4 AS A MARKER OF\nTUMOR PROGRESSIONCXCR4\nexpression has been associated with disease progression, increased recurrence,\nand reduced survival in many cancer types, as listed in Table 1. As pointed out\nearlier, CXCR4 protein expression is detectable in the majority of cases of DCIS\nof the breast, whereas CXCR4 levels are very low in adjacent normal breast\nepithelium [46]. This suggests that the acquisition\nof CXCR4 expression may occur very early in malignant transformation, suggesting\nits potential as a biomarker. As indicated earlier, it has been suggested that\nCXCR4 expression may be useful as an indicator of prognosis [56, 73].Although\nmutations in the CXCR4 gene have not\nbeen reported in the context of cancer, patients with a single nucleotide\npolymorphism in the 3′ untranslated region of the CXCL12 gene had reduced incidence of long distance metastasis of\nepidermoid non-small cell lung cancer (NSCLC) [108].10. PRECLINICAL EFFICACY OF\nANTI-CXCR4 TREATMENTSSeveral studies have\ndemonstrated the efficacy of strategies designed to reduce CXCR4 expression or\ninhibit its activity in preclinical models of cancer development and metastasis. A neutralizing anti-CXCR4 antibody prevented\nmetastasis of MDA-MB-231 breast cancer cells in mice [25] and in another study reduced\ntumor growth after intraperitoneal (IP) injection of Namalwa non-Hodgkin's lymphoma cells [86]. Interestingly, a neutralizing antibody\nagainst CXCR4 also inhibited the growth of subcutaneous tumors derived from\npancreatic cancer cells that did not themselves express CXCR4, probably because\nof the ability of the antibody to block CXCR4 on tumor vasculature [109].CXCR4 peptide\nantagonists have also proven effective in preclinical cancer models. The CXCR4 peptide antagonist 4F-benzoyl-TN14003\ninhibited lung metastasis of MDA-MB-231 breast cancer cells [110], and\n4F-benzoyl-TE14011 reduced pulmonary metastasis of B16-BL6 melanoma cells [111]. Murakami and colleagues assessed the\ncontribution of CXCR4 to the metastatic process by transducing B16 murine\nmelanoma cells with CXCR4, followed by IV injection in syngeneic B57BL/6 mice [112]. CXCR4 expression in this context led to\nincreased pulmonary metastasis, which was reduced with the CXCR4 peptide\nantagonist T22. Liang and colleagues showed that TN14003 itself, which is a 14-mer peptide CXCR4 antagonist,\ninhibited in vitro invasion of\nMDA-MB-231 breast cancer cells and lung metastasis after tail vein injection of\nthese cells, without causing any toxicity [113].Small molecule (nonpeptide)\ninhibitors of CXCR4 have also been tested in preclinical cancer models. Rubin and colleagues showed that the\nnoncompetitive CXCR4 antagonist AMD3100 inhibited tumor growth after\nintracranial implantation of Daoy medulloblastoma cells and U87 glioblastoma\ncells [63] and also inhibited peritoneal carcinomatosis and ascites formation\nafter IP inoculation of NUGC4 human gastric carcinoma cells [78]. In a different approach, blocking the\nmammalian target of rapamycin (mTOR) pathway downstream of CXCR4 was shown to\nsuppress processes involved in the peritoneal dissemination of gastric cancer [114].Liang and\ncolleagues also showed the preclinical efficacy of anti-CXCR4 treatments using\nan RNA-silencing molecular approach [115]. \nMDA-MB-231 breast cancer cells transfected with siRNA oligonucleotides\nto knock down CXCR4 were injected into the tail veins of SCID mice. Mice received twice-weekly IV injections of\nsiRNA oligonucleotides to maintain CXCR4 knockdown. The control mice all developed lung metastases,\nwhereas only one of six mice receiving CXCR4 siRNA-transfected cells and\nfollowup injections with CXCR4 siRNA developed metastases. Stable knockdown of\nCXCR4 expression in 4T1 murine breast carcinoma cells using short hairpin RNA\nreduced orthotopic tumor growth and lung metastasis [49]. Similarly, MDA-MB-231\ncells that had undergone stable knockdown of CXCR4 did not form tumors or lung\nmetastases after orthotopic injection into mammary fat pads of SCID mice,\nwhereas CXCR4-positive cells were tumorigenic [116]. NSCLC 95D lung cancer cells in which CXCR4\nwas knocked down using antisense technology also formed lung metastases in\nfewer mice after SC injection compared to CXCR4 positive cells [88]. Finally, manipulations of CXCR4 expression\nhave become possible using microRNAs (miRNAs), which are endogenous short RNAs\nwith the ability to repress the translation of target mRNAs [117–119]. The\napproach of expressing a synthetic miRNA against CXCR4 mRNA to knock down CXCR4\nexpression has been used successfully in MDA-MB-231 breast cancer cells, HeLa\ncervical carcinoma cells, and U2-OS osteosarcoma cells [118, 120, 121]. Reduced\nCXCR4 expression in the breast cancer model was accompanied by reduced\nmigration and invasion of the cells in\nvitro and fewer lung metastases in\nvivo [121]. These studies show the importance of CXCR4 expression in\nboth primary and secondary tumor growth.11. CLINICAL ASSESSMENT OF\nCXCR4-TARGETED REAGENTSThe bicyclam\ncompound AMD3100 was developed as a small molecule CXCR4 antagonist [122]. Although this compound has not yet been fully\nassessed in clinical trials to determine its therapeutic potential in cancer, it\nhas been examined in small trials in the context of HIV treatment and\nhematopoietic progenitor cell mobilization [123–128]. One trial with AMD3100 reported one patient\nwith thrombocytopenia, two patients with premature ventricular contractions,\nand several patients with paresthesias [126]. \nAMD3100 did not reduce viral load in HIV patients [122], but did\neffectively increase hematopoietic progenitor cell mobilization [124, 125, 127, 128]. However, the mechanisms of action are under\ndebate and may be unrelated to inhibition of CXCR4 as was first presumed.12. REGULATION OF CXCR4 EXPRESSION BY\nFACTORS WITHIN THE TUMORZeelenberg and\ncolleagues found that CT-26 murine colon carcinoma cells grown in vitro expressed CXCR4 mRNA, but\ncell-surface protein levels were not detectable [71]. When the same cells were freshly isolated\nfrom lung or liver metastases or from intrasplenic tumors, cell-surface\nexpression was strongly upregulated. \nThis elevated expression was lost after 2–4 days in culture, indicating\nthat it was not due to selection of a subpopulation of cells with a high CXCR4\nexpression. The authors concluded that\nCXCR4 expression was induced by the in\nvivo tumor microenvironment. Although\nothers have shown that metastatic cells maintain high CXCR4 expression when\ncultured in vitro [129], and\nindeed CXCR4 has been suggested as a cancer stem cell biomarker [130], as\ndiscussed below there is substantial evidence indicating that CXCR4\nexpression is nevertheless influenced by the tumor microenvironment. Additionally,\naberrant activation of signaling pathways within cancer cells, such as those\ninitiated through HER2, can also contribute to elevated CXCR4 expression [131].Multiple features\nand factors present in the tumor microenvironment have been shown to regulate\nCXCR4 expression on tumor cells and other cell types. One such feature is hypoxia [97, 132]. Solid tumors tend to be hypoxic due to\nstructural abnormalities in their vasculature [133]. Staller and colleagues were the first to\ndemonstrate the involvement of hypoxia in the regulation of CXCR4 expression [97]. Their goal was to identify genes regulated by\nthe von Hippel-Lindau tumor suppressor protein\n(pVHL) in renal cell carcinoma cells. pVHL is often inactivated in renal cell\ncancer (RCC) leading to constitutive activation of hypoxia-inducible factor-1\n(HIF-1) target genes. In a microarray\nanalysis, they found that CXCR4 mRNA expression was suppressed by the reintroduction of functional pVHL into\npVHL-deficient A498 RCC cells, an effect that was due to inactivation of HIF-1. CXCR4 protein was also downregulated,\nresulting in reduced migration of RCC cells towards CXCL12. Hypoxia increased CXCR4 mRNA expression in\nHEK-293 human embryonic kidney cells and primary human proximal renal tubular\nepithelial cells, and a hypoxia response element (HRE) was identified within the\nCXCR4 promoter [97]. The authors\nspeculated that intratumoral hypoxia may lead to increased CXCR4 expression in\ndiverse types of solid tumors, increasing metastasis to distant organs. Shioppa and colleagues found that hypoxia\nincreased CXCR4 mRNA and cell-surface protein expression in several cell types,\nincluding monocytes, human monocyte-derived macrophages, tumor-associated\nmacrophages, HUVECs, CAOV3 ovarian carcinoma cells, and MCF-7 breast carcinoma\ncells, leading to increased migration towards CXCL12 due to the activation of\nHIF-1 [132].The hypoxic environment within tumors also\nleads to high extracellular levels of adenosine (adenine-9-β-D-ribofuranoside),\na nucleoside that is involved in energy metabolism and comprises the core\nstructure for adenine nucleotides. The concentration of adenosine in the\nextracellular fluid of solid tumors is about 100-fold that of adjacent normal\ntissue [134]. Adenosine concentrations in tumors reach levels that can act on\nany of four subtypes of adenosine-selective, G-protein-coupled receptors: A1,\nA2a, A2b, and A3 [135]. Adenosine receptors of all four known subtypes are\nexpressed differentially on different cell types within the tumor, including\nstromal cells, endothelial cells, and infiltrating leukocytes. We have shown\nthat through such receptors, adenosine can have protumor effects directly on\ncancer cells and also indirectly via other\nsupporting/infiltrating cells [136–139]. Adenosine also acts through A2a and\nA2b adenosine receptors on human colorectal carcinoma cells to upregulate CXCR4\nmRNA expression up to 10-fold, and selectively increase cell-surface CXCR4\nprotein up to 3-fold [31]. This increase in cell-surface CXCR4 enables the\ncarcinoma cells to migrate toward CXCL12 and enhances their proliferation in\nresponse to CXCL12.One of the further\nmajor factors that allows tumor expansion is vascular endothelial growth factor\n(VEGF), which is also produced in response to hypoxia and which promotes\nneovascularisation of the tumor. The angiogenic effect of VEGF increases the\nsupply of nutrients and blood-borne growth factors to allow growth of the\ntumor. There is significant interplay between the roles of VEGF and CXCR4 in\ntumor expansion. Concomitan high expression of CXCR4 and VEGF has been\nobserved in colorectal [74, 75], breast [38], and ovarian [34] cancers, as well\nas in glioma [140] and osteosarcoma [91], in each of which it has been linked\nto increased angiogenesis, invasion, and/or metastasis. Clinical studies have\nshown that although VEGF and CXCR4 both predispose to lymphatic involvement and\nnodal metastasis in colorectal cancer, they work through different regulatory\nstrategies [74]. Their collaborative role in angiogenesis parallels a similar\njoint action in noncancer processes involving neovascularisation (e.g., [141]), and it has been suggested in\nthe context of tumor angiogenesis that their actions may be synergistic [34].\nIt is not surprising that these two entities are closely linked; VEGF receptors\nand CXCR4 have common regulatory pathways. For example, interference with Notch\nsignalling leads to downregulation of both VEGF receptor 2 and CXCR4 [142].The\nrelationship between VEGF and CXCR4 is complex. Firstly, VEGF can promote CXCR4\npathways. VEGF is present in high levels in tumors and may upregulate CXCR4\nexpression on tumor cells, as has been demonstrated in glioma [143] and breast\ncancer [144]. In the case of tumor cells, this upregulation of CXCR4 by VEGF\ncan happen through an autocrine mechanism [144]. VEGF can also upregulate CXCR4\non the endothelial cells that may be involved in angiogenesis during tumor\nexpansion [145, 146].Conversely,\nthe ability of CXCR4 to signal through PI3K/Akt and ERK1/2 provides a route\nthrough which VEGF expression may be regulated by CXCR4 [147–149]. Binding of CXCL12 to CXCR4 has been shown to\nincrease cellular secretion of VEGF in ovarian cancer [150], breast cancer [147],\nprostate cancer [149, 151], and malignant glioma [152]. This phenomenon\nparallels the ability of the CXCL12/CXCR4 axis to stimulate VEGF secretion in\nnormal lymphohematopoietic cells [153]. One might therefore expect a large part of the antitumor activity of\nCXCR4 antagonists to be mediated through reduced secretion of VEGF. Indeed,\ninterference with the CXCL12-CXCR4 pathway has been shown to cause\ndownregulation of expression of VEGF [39]. However, blocking the CXCL12/CXCR4\naxis in vivo can inhibit tumor\ngrowth and angiogenesis without producing alterations in VEGF pathways [109].Other\ngrowth factors whose levels are elevated in tumors may also enhance\nCXCR4-dependent mechanisms. Tumors have high levels of tumor necrosis factor-α\n(TNF-α), derived primarily from tumor-associated macrophages (TAMs) [154–156]. TNF-α itself, or macrophages that serve as a source of TNF-α, are\nable to increase CXCR4 mRNA and cell-surface protein expression on ovarian\ncancer cells [157] and astroglioma cells [158]. A significant correlation\nbetween TNF-α and CXCR4 expression was found in ovarian cancer biopsies [157]. The increase in CXCR4 at a cellular level\nappears to be due to TNF-α-induced activation of NF-κB signaling and is\nassociated with enhanced migration towards CXCL12 [157]. Therefore, TAMs may contribute to increased\nCXCR4 expression on cancer cells via\nproduction of TNF-α.Finally,\npolypeptide growth factors that are associated with the extracellular matrix,\nand indeed components of the extracellular matrix itself, can upregulate CXCR4\non cancer cells. Transforming growth\nfactor-β (TGF-β) increases cell-surface CXCR4 protein expression on human\nmelanoma cells [35] and we have recently found that FGF-2 upregulates CXCR4 on\nhuman colorectal cancer cells (Bseso B and Blay J, manuscript in preparation). Furthermore, type-I collagen and the\npreparation Matrigel, which is a secreted ECM rich in laminin [159], also\nincrease levels of CXCR4 on melanoma cells [35]. Therefore, interactions with matrix proteins\nwithin tumors may also increase CXCR4 expression.13. THE ROLE OF CYCLOOXYGENASE-2\nAND PGE2 IN CANCER The shift to\nmalignancy in epithelia and indeed the progression to invasion and metastasis\nare associated with increased expression of the enzyme cyclooxygenase-2 (COX-2)\n[160–163]. High COX-2 expression is in\ncancer is often associated with reduced patient survival [163]. The immediate\neffect of high COX-2 expression is increased prostaglandin synthesis, particularly\nprostaglandin E2 (PGE2) [164], which in experimental\nmodels is associated with the production of vascular loops and arches and\nevidence of abnormal vessel function [165], a phenotype consistent with tumor\nangiogenesis. Observations of increased\nexpression of angiogenic regulatory genes, including VEGF, ang-1, and ang-2 are\nconsistent with this view [166]. Furthermore, nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit\ncyclooxygenases, reduce both tumor incidence and microvessel density in\nCOX-2-expressing mice [166] and reduce cancer progression in preclinical models\nand clinical trials [167]. Indeed, NSAIDs\nand COX-2 inhibitors reduce the relative risk of developing colorectal cancer\nby 40–50% [167–169].Tumor-promoting\neffects of COX-2 overexpression appear to be due in large part to increased PGE2 production [170–173]. Associated with the increase in COX-2, there is a\ndecreased expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an\nenzyme involved in the inactivation of PGE2, in cancer compared to normal tissues [174], as well as\nupregulation of cytosolic PLA2 (cPLA2), which increases the supply of arachidonic acid substrate\nfor COX-2 [175–177]. In addition to promoting angiogenesis, PGE2 also stimulates cancer cell proliferation [178, 179], promotes cell migration [180],\nand causes transactivation of polypeptide growth factor receptors [181].14. OTHER PROSTAGLANDINS IN CANCERProstaglandins\ntogether with the thromboxanes are classed as prostanoids, and belong to a larger group of compounds\nreferred to as eicosanoids [182]. The main prostanoids apart from PGE2 are prostaglandin F2α (PGF2α), prostaglandin D2 (PGD2), prostaglandin I2 (PGI2 or\nprostacyclin), and thromboxane A2 (TXA2). As well as\nreflecting changes in COX-2, cPLA2, and inactivating enzymes, the\nlevels of different prostanoids in tumors can be modulated by altered expression\nof specific prostaglandin synthases [183]. Prostaglandins can also be metabolized\nnonenzymatically to form a range of products both in the body and in cell\nculture. PGD2 can be\nconverted to cyclopentenone J-series prostaglandins, including prostaglandin J2 (PGJ2), 9-deoxy-Δ9,Δ12‐13,14-dihydro-PGD2 (Δ12-PGJ2), and 15-deoxy-Δ12,14-PGJ2 (15dPGJ2); PGE2 can be converted to prostaglandin A2 (PGA2) [184–186]. The tumor microenvironment therefore has a rich\nand varied content of eicosanoid mediators.15. PROSTAGLANDIN EFFECTS ON CANCER CELLSAlthough the major\nfocus of attention has been on PGE2, a range of eicosanoids acts to\nrestrain tumor growth. Indeed the PGE2 metabolite PGA2 reduces\ncell number and induces apoptosis and cell cycle changes in both human breast\ncancer cells and human epithelial cervical carcinoma cells [187].More notably, PGD2 and its series of derivatives have anticancer effects. PGD2 itself can reduce the growth of \ncarcinoma cells [188]. However, other studies have shown that the nonenzymatic\nbreakdown of PGD2 to sequential metabolites (Figure 1) may be required\nfor growth inhibition and that the latter metabolites are the active\neicosanoids [189–194]. PGD2 therefore can act independently of its DP\nreceptors by its metabolism through a dehydration reaction to prostaglandin J2 (PGJ2), Δ12-PGJ2, and then to\n15-deoxy-Δ12,14-prostaglandin J2(15dPGJ2) [184].\nThis reaction occurs in cell culture media, both in the presence and absence of\nserum [184, 189, 195]. Therefore, it is\npossible that many effects noted in\nvitro with PGD2 are actually due to the formation of J-series\nprostaglandins. Frequent replacement\nwith fresh medium containing PGD2 in such circumstances can\neliminate the response, while the addition of the metabolite(s) themselves\nleads to growth inhibition in a shorter timeframe than PGD2 itself [189].\nSome workers have proposed that Δ12-PGJ2 is the key\nmetabolite [189]; but in fact all of the successive J-series prostaglandins,\nthat is, PGJ2, Δ12-PGJ2, and 15dPGJ2,\nare able to reduce proliferation and induce apoptosis of cancer cells [190]. Furthermore, the end metabolite 15dPGJ2 is active against many cell types, including colorectal carcinoma cells [191, 192],\nprostate carcinoma cells [193], and Burkitt lymphoma cells [194], suggesting\nthat 15dPGJ2 may be the crucial mediator.16. THE ROLE OF 15dPGJ2 AND ITS ACTION ON PPARγ\n15dPGJ2 is an agonist for the nuclear receptor peroxisome proliferator-activated\nreceptor γ (PPARγ) [196, 197], and activation of PPARγ may account for the\ngrowth inhibitory effects of 15dPGJ2. PPARγ activation results in\nits heterodimerization with the retinoid X receptor (RXR), binding to\nperoxisome proliferator response elements (PPREs) on DNA, and subsequent\nactivation of target gene expression [198]. \nPPARγ is aberrantly expressed in some cancer types [199], and in many\ncases its activation leads to cell death or differentiation [191, 200, 201]. This\naction of 15dPGJ2, and by extension its precursors PGD2,\nPGJ2, and Δ12-PGJ2, may underlie the\nmajor action of these eicosanoids on cell growth. For example, 15dPGJ2 reduces the growth of PC-3 human prostate cancer cells through the activation\nof PPARγ [202]. However, in addition to\ndirect growth-inhibitory effects, 15dPGJ2 may also exert anticancer\neffects by reducing expression of protumor proteins. For example, 15dPGJ2 inhibits\nphorbol ester-induced VEGF and COX-2 expression in SW620 human colorectal\ncarcinoma cells [203].17. 15dPGJ2 CAUSES DOWNREGULATION OF\nCXCR4 ON CANCER CELLSIn\nour studies of the possible effects of these different prostaglandins on CXCR4,\nwe focused upon the expression of the mature protein and furthermore restricted\nour quantitation exclusively to the receptor that is displayed to the external\nenvironment at the cell surface [31]. Cell-surface CXCR4 reflects functional\nreceptor that is coupled to cellular responses [31] rather than the very large\nintracellular pool of inaccessible receptor protein [72].Although\nPGF2α (to some extent) and PGE2 (as well as its product\nPGA2) have some ability to modulate CXCR4 levels, by far the most\npotent prostaglandins in this regard are PGD2 and its derivatives [204]. Prostaglandin D2 and the J-series\nprostaglandins used at low micromolar concentrations cause substantial loss of\nCXCR4 from the surface of HT-29 human colorectal carcinoma cells [204]. In particular, 15dPGJ2 completely\neliminates cell-surface CXCR4 at a concentration of 10−5 M in vitro, and has significant effects\nafter a single dose of 300 nM, about 100-fold less than for PGF2α [204].\nThe time course of the decline in cell-surface CXCR4 protein is slow, reaching\na maximum only after 48–72 hours (Figure 2). The concentrations of prostaglandins\nthat are needed to cause downregulation after a single dose likely grossly\noverestimate the steady-state levels that would cause such a response, as we\nhave found in other studies with labile metabolites [31, 138]. We estimate that the\neffect of 15dPGJ2 on CXCR4 is achievable with concentrations of\n15dPGJ2 present in vivo.As\ncan be seen in Figure 2, the response to 15dPGJ2 occurs more rapidly\nthan that to PGJ2, which in turn has a more rapid onset than PGD2.\nWe further found that each of these prostaglandins does suppress CXCR4 mRNA\nexpression and that the effect of 15dPGJ2 again occurs earlier than\nthat of PGD2 [204]. The different relative kinetics of the\ndownregulation of CXCR4 for the J-series prostaglandins are consistent with\ndata on the conversion of PGD2 through to 15dPGJ2 [189] pointing\nto 15dPGJ2 as the key factor in controlling the levels of functional\nCXCR4. PGD2 produces similar downregulation of CXCR4 in other cell\ntypes such as the T47D human breast carcinoma cell line (Richard CL, Blay J,\nunpublished observations), suggesting that this may be a common phenomenon. The\ndownregulation of CXCR4 expression by 15dPGJ2 differs from 15 dPGJ2-mediated\ndownregulation of other proteins, including cyclin D1 and estrogen receptor α,\nwhich has been shown to occur through protein degradation rather than through\nchanges in transcription [205].18. 15dPGJ2 DOWNREGULATES CXCR4\nPRIMARILY VIA PPARγ\nThe main target\nfor 15dPGJ2 is the nuclear receptor PPARγ [196, 197]. We found that\nthe ability of 15dPGJ2 to downregulate CXCR4 occurred primarily\nthrough this pathway. The effect of 15dPGJ2 was mimicked by PPARγ\nagonists such as rosiglitazone (Table 2, [206]), and antagonized or blocked by\nthe PPARγ antagonists GW9662 and T0070907 [204], which are irreversible\ninhibitors of PPARγ [207, 208]. A minor part of the downregulatory activity of\n15dPGJ2 was due to the inhibition of NFκB since the 15dPGJ2 analogue CAY10410 (9,10-dihydro-15-deoxy-Δ12,14-prostaglandin J2)\n[209, 210], which retains the ability to act on PPARγ but lacks the ability of\n15dPGJ2 to inhibit NFκB, was less potent than 15dPGJ2 [208]. It is the cyclopentenone structure of 15dPGJ2 (not present in CAY10410) that confers an ability to inhibit NFκB [211].\nConsistent with a role for this structure, cyclopentenone itself (but not\ncyclopentane or cyclopentene) caused downregulation of CXCR4 [204]. Furthermore,\nsince PGA2 possesses the cyclopentenone configuration [212], this\nexplains the ability of PGA2 (and that of PGE2) to\ndownregulate CXCR4, although it does not contain the α,β-unsaturated ketone\nmoiety necessary to activate PPARγ signaling [210].The\nexistence of a mechanism of 15dPGJ2-induced CXCR4 downregulation\nmay, in evolutionary terms, be an extension of the anti-inflammatory effects of\n15dPGJ2. Late in the inflammation process the prostaglandin profile\nshifts from a PGE2-rich state to a PGD2-rich (and\ntherefore 15dPGJ2-rich) state, leading to the resolution of\ninflammation [213]. Reduced CXCR4\nexpression may be an additional mechanism by which 15dPGJ2 attempts\nthe resolution of inflammation.It\nis clear that this mechanism is not operative in the context of metastatic\ntumors, because CXCR4 levels are characteristically high (Table 1). Unlike PGE2 which is present in elevated concentration in tumors [170–173], 15dPGJ2 levels are likely low in tumors compared to normal tissue. Levels of its precursor\nPGD2 are low in tissues of familial adenomatous polyposis, a\ncondition that predisposes to colorectal cancer [172], and have been negatively\ncorrelated with hepatic metastasis in tumor tissues taken from patients with\ncolorectal cancer [188]. The enzyme involved in PGD2 synthesis, PGD\nsynthase (PGDS), is decreased in cerebrospinal fluid of brain cancer patients\ncompared to patients without disease [214]. There is a contested report of\nlevels of 15dPGJ2 being decreased during breast cancer progression,\nwith the lowest levels being detected in metastatic disease [173]. Finally,\nmechanisms to sequester or eliminate 15dPGJ2 may be upregulated in\ncancer [215, 216]. Overall, it seems that the predominant prostaglandin within tumors\nis PGE2, and 15dPGJ2 may not be present in high levels at\nall. Thus, 15dPGJ2-dependent suppression of CXCR4 seems to be a\nrestraint mechanism that is not operative in a cancer situation.19. SYNTHETIC PPARγ AGONISTS DOWNREGULATE\nCXCR4 ON CANCER CELLSAs indicated\nabove, the PPARγ agonist rosiglitazone also decreased CXCR4 expression on human\ncolorectal cancer cells, congruent with an effect of 15dPGJ2 through\nPPARγ. This effect was seen at both the mRNA and protein level, and was more\ndurable than the effect of 15dPGJ2, as it would be expected for a\nmore chemically stable ligand [101, 204]. Moreover, we found that other\nglitazone agents also downregulate CXCR4, with a rank order of potency\n(rosiglitazone > pioglitazone > ciglitazone > troglitazone) consistent\nwith their potencies for interaction with PPARγ [206, 217, 218]. Further confirming that these agents were\nacting through their expected target, PPARγ, and that this target is linked to\nelimination or reduction of CXCR4 at the cell surface, we showed that the\nability of rosiglitazone to decrease CXCR4 was blocked by the PPARγ antagonists\nGW9662 and T0070907 (Table 2), or by shRNA knockdown of PPARγ expression in the\ncancer cells [101].Therefore,\nrosiglitazone and its analogues act through PPARγ to cause substantial and\npersistent suppression of CXCR4 on cancer cells. Since these agents are the\nsame chemicals as the thiazolidinedione (TZD) class of drugs that have been\nused clinically for the treatment of diabetes (although recent concerns\nregarding side effects have limited their utility), it opens up the possibility\nthat we may already have a means to manipulate CXCR4 levels in cancer. Given\nthat CXCR4 expression is linked to metastasis, judicious use of TZDs may allow\nus an opportunity to influence the metastatic process (Figure 3). 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ChangS-HLiuCHConwayRRole of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progressionProceedings of the National Academy of Sciences of the United States of America2004101259159614688410\n166. ChangS-HAiYBreyerRMLaneTFHlaTThe prostaglandin E2 receptor EP2 is required for cyclooxygenase 2-mediated mammary hyperplasiaCancer Research200565114496449915930264\n167. GuptaRADuBoisRNColorectal cancer prevention and treatment by inhibition of cyclooxygenase-2Nature Reviews Cancer200111112111900248\n168. MarnettLJDuBoisRNCOX-2: a target for colon cancer preventionAnnual Review of Pharmacology and Toxicology2002425580\n169. WangDMannJRDuBoisRNThe role of prostaglandins and other eicosanoids in the gastrointestinal tractGastroenterology200512851445146115887126\n170. RigasBGoldmanISLevineLAltered eicosanoid levels in human colon cancerJournal of Laboratory and Clinical Medicine199312255185238228569\n171. PughSThomasGAPatients with adenomatous polyps and carcinomas have increased colonic mucosal prostaglandin E2\nGut19943556756788200564\n172. DuBoisRNHylindLMRobinsonCRProstaglandin levels in human colorectal mucosa: effects of sulindac in patients with familial adenomatous polyposisDigestive Diseases and Sciences19984323113169512123\n173. BadawiAFBadrMZExpression of cyclooxygenase-2 and peroxisome proliferator-activated receptor-γ and levels of prostaglandin E2 and 15-deoxy-Δ12,14-prostaglandin J2 in human breast cancer and metastasisInternational Journal of Cancer20031031849012455057\n174. BacklundMGMannJRHollaVR15-hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancerThe Journal of Biological Chemistry200528053217322315542609\n175. SoydanASTavaresIAWeechPKTremblayNMBennettAHigh molecular weight phospholipase A2 and fatty acids in human colon tumours and associated normal tissueEuropean Journal of Cancer1996321017811787\n176. DimbergJSamuelssonAHuganderASöderkvistPGene expression of cyclooxygenase-2 group II and cytosolic phospholipase A2 in human colorectal cancerAnticancer Research1998185A328332879858896\n177. ÖsterströmADimbergJFransénKSöderkvistPExpression of cytosolic and group X secretory phospholipase A2 genes in human colorectal adenocarcinomasCancer Letters2002182217518212048163\n178. QiaoLKozoniVTsiouliasGJSelected eicosanoids increase the proliferation rate of human colon carcinoma cell lines and mouse colonocytes in vivoBiochimica et Biophysica Acta1995125822152237548186\n179. WangDBuchananFGWangHDeySKDuBoisRNProstaglandin E2 enhances intestinal adenoma growth via activation of the Ras-mitogen-activated protein kinase cascadeCancer Research20056551822182915753380\n180. TimoshenkoAVXuGChakrabartiSLalaPKChakrabortyCRole of prostaglandin E2 receptors in migration of murine and human breast cancer cellsExperimental Cell Research2003289226527414499627\n181. PaiRSoreghanBSzaboILPavelkaMBaatarDTarnawskiASProstaglandin E2, transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophyNature Medicine200283289293\n182. FunkCDProstaglandins and leukotrienes: advances in eicosanoid biologyScience200129455481871187511729303\n183. ErmertLDierkesCErmertMImmunohistochemical expression of cyclooxygenase isoenzymes and downstream enzymes in human lung tumorsClinical Cancer Research2003951604161012738712\n184. FitzpatrickFAWynaldaMAAlbumin-catalyzed metabolism of prostaglandin D2. Identification of products formed in vitroThe Journal of Biological Chemistry19832581911713117186578214\n185. AusselCMaryDFehlmannMProstaglandin synthesis in human T cells: its partial inhibition by lectins and anti-CD3 antibodies as a possible step in T cell activationThe Journal of Immunology198713810309430993106472\n186. IshiharaOSullivanMHFElderMGDifferences of metabolism of prostaglandin E2 and F2α by decidual stromal cells and macrophages in cultureEicosanoids1991442032071789996\n187. JoubertAMPanzerABianchiPCLotteringM-LThe effects of prostaglandin A2 on cell growth, cell cycle status and apoptosis induction in HeLa and MCF-7 cellsCancer Letters2003191220320912618334\n188. YoshidaTOhkiSKanazawaMInhibitory effects of prostaglandin D2 against the proliferation of human colon cancer cell lines and hepatic metastasis from colorectal cancerSurgery Today19982877407459697269\n189. NarumiyaSFukushimaM\nΔ12-prostaglandin J2, an ultimate metabolite of prostaglandin D2 exerting cell growth inhibitionBiochemical and Biophysical Research Communications198512737397453857041\n190. ClayCENamenAMAtsumiG-IInfluence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cellsCarcinogenesis199920101905191110506103\n191. KitamuraSMiyazakiYShinomuraYKondoSKanayamaSMatsuzawaYPeroxisome proliferator-activated receptor γ induces growth arrest and differentiation markers of human colon cancer cellsJapanese Journal of Cancer Research1999901758010076568\n192. ShimadaTKojimaKYoshiuraKHiraishiHTeranoACharacteristics of the peroxisome proliferator activated receptor γ (PPARγ) ligand induced apoptosis in colon cancer cellsGut200250565866411950812\n193. MuellerESmithMSarrafPEffects of ligand activation of peroxisome proliferator-activated receptor γ in human prostate cancerProceedings of the National Academy of Sciences of the United States of America20009720109901099510984506\n194. PivaRGianferrettiPCiucciATaulliRBelardoGSantoroMG15-deoxy-Δ12,14-prostaglandin J2 induces apoptosis in human malignant B cells: an effect associated with inhibition of NF-κB activity and down-regulation of antiapoptotic proteinsBlood200510541750175815498850\n195. ShibataTKondoMOsawaTShibataNKobayashiMUchidaK15-deoxy-Δ12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processesThe Journal of Biological Chemistry200227712104591046611786541\n196. FormanBMTontonozPChenJBrunRPSpiegelmanBMEvansRM15-deoxy-Δ12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARγ\nCell19958358038128521497\n197. KliewerSALenhardJMWillsonTMPatelIMorrisDCLehmannJMA prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor γ and promotes adipocyte differentiationCell19958358138198521498\n198. KotaBPHuangTH-WRoufogalisBDAn overview on biological mechanisms of PPARsPharmacological Research2005512859415629253\n199. DuBoisRNGuptaRBrockmanJReddyBSKrakowSLLazarMAThe nuclear eicosanoid receptor, PPARγ, is aberrantly expressed in colonic cancersCarcinogenesis199819149539472692\n200. BrockmanJAGuptaRADuBoisRNActivation of PPARγ leads to inhibition of anchorage-independent growth of human colorectal cancer cellsGastroenterology19981155104910559797355\n201. ElstnerEMüllerCKoshizukaKLigands for peroxisome proliferator-activated receptory and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX miceProceedings of the National Academy of Sciences of the United States of America19989515880688119671760\n202. KimJYangPSuraokarMSuppression of prostate tumor cell growth by stromal cell prostaglandin D synthase-derived productsCancer Research200565146189619816024620\n203. GrauRIñiguezMAFresnoMInhibition of activator protein 1 activation, vascular endothelial growth factor, and cyclooxygenase-2 expression by 15-deoxy-Δ12,14-prostaglandin J2 in colon carcinoma cells: evidence for a redox-sensitive peroxisome \nproliferator-activated receptor-γ-independent mechanismCancer Research200464155162517115289320\n204. RichardCLLowthersELBlayJ15-deoxy-Δ12,14-prostaglandin J2 down-regulates CXCR4 on carcinoma cells through PPARγ- and NFκB-mediated pathwaysExperimental Cell Research2007313163446345817707368\n205. QinCBurghardtRSmithRWormkeMStewartJSafeSPeroxisome proliferator-activated receptor γ agonists induce proteasome-dependent degradation of cyclin D1 and estrogen receptor α in MCF-7 breast cancer cellsCancer Research200363595896412615709\n206. LehmannJMMooreLBSmith-OliverTAWilkisonWOWillsonTMKliewerSAAn antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ)The Journal of Biological Chemistry19952702212953129567768881\n207. LeeGElwoodFMcNallyJT0070907, a selective ligand for peroxisome proliferator-activated receptor γ, functions as an antagonist of biochemical and cellular activitiesThe Journal of Biological Chemistry200227722196491965711877444\n208. LeesnitzerLMParksDJBledsoeRKFunctional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662Biochemistry200241216640665012022867\n209. LindströmTMBennettPR15-deoxy-Δ12,14-prostaglandin J2 inhibits interleukin-1β-induced nuclear factor-κB in human amnion and myometrial cells: mechanisms and implicationsThe Journal of Clinical Endocrinology & Metabolism20059063534354315755849\n210. ShirakiTKamiyaNShikiSKodamaTSKakizukaAJingamiH\nα,β-unsaturated ketone is a core moiety of natural ligands for covalent binding to peroxisome proliferator-activated receptor γ\nThe Journal of Biological Chemistry200528014141451415315695504\n211. StrausDSPascualGLiM15-deoxy-Δ12,14-prostaglandin J2 inhibits multiple steps in the NF-κB signaling pathwayProceedings of the National Academy of Sciences of the United States of America20009794844484910781090\n212. RossiAKapahiPNatoliGAnti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IκB kinaseNature2000403676510311810638762\n213. SchuligoiRGrillMHeinemannAPeskarBAAmannRSequential induction of prostaglandin E and D synthases in inflammationBiochemical and Biophysical Research Communications2005335368468916084489\n214. SasoLLeoneMGSorrentinoCQuantification of prostaglandin D synthetase in cerebrospinal fluid: a potential marker for brain tumorBiochemistry & Molecular Biology International19984646436569844724\n215. PaumiCMWrightMTownsendAJMorrowCSMultidrug resistance protein (MRP) 1 and MRP3 attenuate cytotoxic and transactivating effects of the cyclopentenone prostaglandin, 15-deoxy-Δ12,14prostaglandin J2 in MCF7 breast cancer cellsBiochemistry200342185429543712731885\n216. PaumiCMSmithermanPKTownsendAJMorrowCSGlutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor γ (PPARγ) ligand, 15-deoxy-Δ12,14prostaglandin J2 (15-d-PGJ2)Biochemistry20044382345235214979731\n217. ReginatoMJBaileySTKrakowSLA potent antidiabetic thiazolidinedione with unique peroxisome proliferator-activated receptor γ-activating propertiesThe Journal of Biological Chemistry19982734932679326849830009\n218. CampHSLiOWiseSCDifferential activation of peroxisome proliferator-activated receptor-γ by troglitazone and rosiglitazoneDiabetes200049453954710871190"
+}

batch_10/PMC2528551.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2528551",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528551\nAUTHORS: M.L.S. Vachon\n\nABSTRACT:\nCancer can lead to spiritual transformation, which can be seen as a form of alchemy. During this process, patients, family members, and even professional caregivers can find themselves having spiritual experiences that go beyond any they had previously encountered. This paper provides qualitative descriptions of the “Field” or “Soul Wisdom” experienced by patients and caregivers.\n\nBODY:\nNo Body Content\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2528896.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2528896",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528896\nAUTHORS: Alastair D Hay, Céire Costelloe, Niamh M Redmond, Alan A Montgomery, Margaret Fletcher, Sandra Hollinghurst, Tim J Peters\n\nABSTRACT:\nObjective To investigate whether paracetamol (acetaminophen) plus ibuprofen are superior to either drug alone for increasing time without fever and the relief of fever associated discomfort in febrile children managed at home.Design Individually randomised, blinded, three arm trial.Setting Primary care and households in England.Participants Children aged between 6 months and 6 years with axillary temperatures of at least 37.8°C and up to 41.0°C.Intervention Advice on physical measures to reduce temperature and the provision of, and advice to give, paracetamol plus ibuprofen, paracetamol alone, or ibuprofen alone.Main outcome measures Primary outcomes were the time without fever (<37.2°C) in the first four hours after the first dose was given and the proportion of children reported as being normal on the discomfort scale at 48 hours. Secondary outcomes were time to first occurrence of normal temperature (fever clearance), time without fever over 24 hours, fever associated symptoms, and adverse effects.Results On an intention to treat basis, paracetamol plus ibuprofen were superior to paracetamol for less time with fever in the first four hours (adjusted difference 55 minutes, 95% confidence interval 33 to 77; P<0.001) and may have been as good as ibuprofen (16 minutes, −7 to 39; P=0.2). For less time with fever over 24 hours, paracetamol plus ibuprofen were superior to paracetamol (4.4 hours, 2.4 to 6.3; P<0.001) and to ibuprofen (2.5 hours, 0.6 to 4.4; P=0.008). Combined therapy cleared fever 23 minutes (2 to 45; P=0.025) faster than paracetamol alone but no faster than ibuprofen alone (−3 minutes, 18 to −24; P=0.8). No benefit was found for discomfort or other symptoms, although power was low for these outcomes. Adverse effects did not differ between groups.Conclusion Parents, nurses, pharmacists, and doctors wanting to use medicines to supplement physical measures to maximise the time that children spend without fever should use ibuprofen first and consider the relative benefits and risks of using paracetamol plus ibuprofen over 24 hours.Trial registration Current Controlled Trials ISRCTN26362730.\n\nBODY:\nIntroductionFever is a normal part of childhood illness, affecting around 70% of preschool children yearly.1 It can be miserable for the child, cause anxiety for parents,2 and be expensive for health services. Up to 40% of preschool children see a health professional for a febrile illness each year.1 Although fever is considered by many to be an advantageous evolutionary byproduct of the host response to infection, and as such should not be treated,3 the use of antipyretics is widespread. The reasons for treating fever are contested and not necessarily evidence based but include minimising discomfort, controlling the fever, and preventing febrile convulsions.Options for treating fever include physical measures (taking cool fluids and dressing lightly) and the antipyretic drugs paracetamol (acetaminophen) and ibuprofen. Evidence for physical measures is now redundant as it mostly pertains to tepid sponging,4 which is no longer recommended.5 Paracetamol and ibuprofen have both been shown to be superior to placebo6\n7\n8 and ibuprofen superior to paracetamol9 for the relief of fever. Given that the drugs have different mechanisms of action10 it is possible that they are more effective together than when used alone, but the evidence to date is sparse and conflicting. Five published trials11\n12\n13\n14\n15 mostly tested the effects of single doses at selected time points (which can arbitrarily advantage one drug because of the difference in times to maximum effect16), were largely done in secondary care, and reached conflicting conclusions. Recently published UK guidelines5 advise the use of either drug (no preference stated) for children with fever who are unwell or distressed and state that owing to the lack of evidence the drugs should not be used together or alternately.We carried out a community based, three arm, blinded, randomised controlled trial to investigate the relative clinical effectiveness of multiple doses (as used for most episodes of fever) of paracetamol plus ibuprofen compared with either drug alone. Our investigation into the relative cost effectiveness is reported in an accompanying paper.17MethodsWe recruited and followed up children between January 2005 and May 2007 using three strategies: local, remote, and community. We invited all NHS organisations providing primary care services in Bristol to assist with recruitment to the trial, including NHS Direct, the walk-in centres, all general practices, the general practitioner out of hours cooperatives, and the emergency department of the Bristol Royal Hospital for Children.During local recruitment the NHS sites invited parents of appropriately aged children to discuss the study with our research nurses, who were present in the waiting rooms. In the remote strategy, clinicians faxed the details of potentially eligible children to the trial administrator, who notified the research nurses. In the community strategy, parents were invited to contact the trial directly by telephone. The telephone number was promoted during local and remote recruitment and in local newspaper and radio advertisements. When parents made contact, the trial administrator notified the research nurses of potentially eligible children.Once aware of potentially eligible children identified through any of the recruitment strategies, research nurses contacted parents by telephone to arrange a meeting (usually at home) to explain the trial fully and to verify eligibility.ParticipantsWe included children if they were aged between 6 months and 6 years and were unwell with a temperature of at least 37.8°C and up to 41.0°C as a result of illnesses that could be managed at home. We excluded children if they required hospital admission; were clinically dehydrated; had recently participated in another trial; had previously participated in PITCH; had a known intolerance, allergy, or contraindication to a trial drug18; had a chronic neurological, cardiac, pulmonary (except asthma), liver, or renal disease; or had parents who could not read or write in English. We followed up children at 24 and 48 hours and at day 5.RandomisationAfter written informed consent had been obtained and the baseline questionnaire completed, the research nurse telephoned a remote, automated randomisation service. Allocation to one of three trial arms (paracetamol plus ibuprofen, paracetamol alone, ibuprofen alone) was minimised19 by age (6-17 months v 18-71 months), severity of fever (37.8°C to 38.9°C v 39.0°C to 41.0°C), discomfort scale (“normal” or “not quite normal” v “some distress” or “very distressed”), previous duration of fever (≤24 hours v >24 hours), and current antibiotic use (yes v no).InterventionParents were given standardised verbal and written advice on the appropriate use of loose clothing and encouraging children to take cool fluids. The intervention was the provision of, and advice to give, the study drugs for up to 48 hours: paracetamol every 4-6 hours (maximum of four doses in 24 hours) and ibuprofen every 6-8 hours (maximum of three doses in 24 hours). Parents, research nurses, and investigators were blinded to treatment allocation by the use of identically matched placebo drugs. All parents received two medicine bottles; either both active or one containing the active drug and the other placebo. Given the differences in dosing, the parents were aware of which was paracetamol/placebo and which was ibuprofen/placebo. All liquid suspensions were sugar-free and supplied in licensed containers with child resistant caps. The dose of drug was determined by the child’s weight: paracetamol 15 mg/kg per dose and ibuprofen 10 mg/kg per dose. At the baseline visit and before randomisation the research nurse weighed the child, undressed to one layer, using scales approved for use in children (Seca, UK). Randomisation was abandoned if weight could not be established and administration of the study drug was deemed unsafe. The research nurse initially calculated the volume of suspension per dose (to the nearest 0.5 ml), which was confirmed during randomisation. The bottles of active drug contained the standard concentrations: 120 mg of paracetamol per 5 ml and 100 mg of ibuprofen per 5 ml.The first doses were given in the presence of the research nurse and were timed to coincide with the child’s next due dose of drug—that is, at least four hours after the last dose of paracetamol and six hours after that of ibuprofen, and were never such that the maximum number of doses over a 24 hour period was exceeded. The order in which the first drug was given was determined randomly. We recorded the time that the drug was swallowed and designated that as time zero. The first four hours, after children were observed to be given the drugs and before any further drug was given, was regarded as the “efficacy period.” We asked the parents to give the drugs regularly from four to 24 hours (“proactive period”). Figure 1 describes the intervention period for the first 24 hours. We asked parents to give the drugs between 24 and 48 hours in response to their child’s symptoms (“reactive period”). At 48 hours we retrieved the study drugs and advised the parents to use over the counter preparations as required until day 5.Fig 1 Use of study drugs during first 24 hours. Shaded areas represent time that drug was to be givenOutcomesWe timed all outcomes in relation to the administration of the first drug doses. The primary outcomes were the number of minutes without fever (<37.2°C) in the first four hours and the proportion of children reported as being normal on the discomfort scale at 48 hours. Secondary outcomes were collected at three time points. In the first 24 hours we recorded the time to temperature first falling below 37.2��C (fever clearance), the time spent without fever over 24 hours, and the proportion of children without fever associated symptoms: discomfort, reduced activity, reduced appetite, and disturbed sleep. At 48 hours and day 5 we obtained data on fever associated symptoms and temperature measured by parents. At all time points we asked parents about adverse effects.We measured time without fever using a technique similar to that in a previous study.20 Using a data logger (OM-CP-RTDTEMP110; Omega Engineering, Stamford, CT) connected to an axillary temperature probe, we measured and recorded temperature every 30 seconds. Parents were asked to help their child keep the logger on for 24 hours. With support from research nurses the parents completed symptom diaries on discomfort, sleep, appetite, and activity using ordered categorical scales. Parents were asked to enter the value best representing their child’s state at the time of recording or in the previous 10 minutes. They also recorded adverse effects (defined as new symptoms or worsening of pre-existing symptoms21) and temperature, which they measured with a standard digital axillary thermometer.Sample sizeIn the original protocol the target difference for the time spent without fever in the first four hours was 30 minutes (with an estimated standard deviation of 80 minutes20) and that for the binary outcome of being rated normal on the discomfort scale at 48 hours was 60% compared with 75% (equivalent to an odds ratio of 2.0). To detect the latter comparison with 90% power and a two sided α of 0.027 (allowing for multiple comparisons between the combined therapy group and each of the two single therapy groups22) we required a total sample size of 747 children. Difficulties with recruitment led to the addition of the remote and community methods and a reduced achievable sample size. For time without fever we estimated a revised standard deviation of 50 minutes on the basis of the first 50 children (independent of allocation group). Along with a revised 80% power, we determined that a total sample size of 180 would allow the detection of the original target difference of 30 minutes. Sensitivity to differences in discomfort was, however, reduced, with odds ratios of only 4 or more being detectable.Statistical analysesWe obtained descriptive statistics to characterise children, assess baseline comparability, and compare side effects. Comparative analyses were done in Stata 9 on an intention to treat basis using linear or logistic regression and adjusting for minimisation variables. Primary comparisons were between paracetamol plus ibuprofen and either drug alone, and secondary comparisons were between paracetamol and ibuprofen, using Dunnett’s and Tukey’s adjustments, respectively, for multiple comparisons.22 For all “time without fever” analyses we regarded as valid only biologically plausible temperatures of more than 33°C and less than 45°C. In regression models we used the proportion of valid time under the fever threshold (with results converted into minutes or hours for presentational purposes) and we weighted these according to the amount of valid data. Secondary analyses included additional adjustment for factors showing possible imbalance at baseline and preplanned exploratory analyses for differential effects of paracetamol plus ibuprofen compared with paracetamol alone or ibuprofen alone for baseline age, temperature, discomfort, antibiotic use, and presence of otitis media. We selected otitis media because affected children might experience enhanced effects for both fever and pain.ResultsThirty five primary care sites in Bristol agreed to take part in the trial: NHS Direct, one walk-in centre, 30 general practices, two general practitioner out of hours cooperatives, and the emergency department of the Bristol Royal Hospital for Children. Figure 2 shows the numbers of children recruited through the three different methods. Overall, 4515 contacts were made, of which 3477 children were ineligible, most commonly (89%) because of insufficient fever. The remaining 1038 children were potentially eligible, but the temperature criterion before randomisation could not be verified in 882 because the parents did not want to commit to the study or had concerns about the drugs (669 declined) or the parents saw a clinician but left without contacting the study team (213 missed). No parent declined at randomisation, and attrition was minimal. Deviations from the protocol occurred; in the first 24 hours (23 hours and 40 minutes), 13 (7) children received an erroneous fifth dose of paracetamol and similarly 18 (13) children an erroneous fourth dose of ibuprofen. In four children, clinicians and parents but not research staff were unblinded to treatment allocation.Fig 2 Participant flow through trialDescriptive resultsThe groups were comparable at baseline, although potentially influential differences existed for sex, method of recruitment, and activity (table 1). Since additional adjustment for these variables had negligible effects in all analyses only minimisation variables were adjusted for in the comparative analyses. Nearly all the children were unwell, with more than 90% experiencing discomfort, reduced activity, abnormal appetite, or abnormal sleep (table 1).Table 1 Baseline characteristics of children with fever randomised to three treatment groups. Values are numbers (percentages) of children unless stated otherwiseCharacteristicParacetamol (n=52)Ibuprofen (n=52)Paracetamol plus ibuprofen (n=52)Boy26 (50)37 (71)25 (48)Girl26 (50)15 (29)27 (52)Mean (SD) weight (kg)13.0 (4.2)13.4 (3.9)12.6 (3.3)Mean (SD) age (months)28.7 (17.7)28.1 (17.4)25.1 (13.4)Age (months)*: 6-1720 (38)18 (35)19 (37) 18-7132 (62)34 (65)33 (63)Mean (SD) baseline temperature (°C)38.6 (0.6)38.6 (0.6)38.6 (0.6)Temperature (°C)*: <3937 (71)37 (71)39 (75) 39-4115 (29)15 (29)13 (25)Fever duration (hours)*: ≤2418 (35)19 (37)19 (37) >2434 (65)33 (63)33 (63)Antibiotic use*: Yes14 (27)15 (29)17 (33) No38 (73)37 (71)35 (67)Paracetamol use 4-6 hours before randomisation: Yes20 (38)17 (33)20 (38) No32 (62)35 (67)32 (62)Ibuprofen use 6-8 hours before randomisation: Yes4 (8)2 (4)3 (6) No48 (92)50 (96)49 (94)Discomfort*: Normal3 (6)5 (9)5 (9) Not quite normal31 (60)27 (52)30 (58) Some pain or distress18 (34)18 (35)14 (27) Crying or very distressed0 (0)2 (4)3 (6)Activity: Normal3 (6)4 (8)4 (8) Quiet for longer than usual12 (23)18 (35)23 (45) Hardly moving about31 (60)19 (36)19 (36) Not moving about willingly6 (11)11 (21)6 (11)Appetite: Normal5 (10)3 (6)4 (8) Eating less than normal12 (23)14 (27)10 (19) Eating much less than normal35 (67)33 (63)36 (69) Vomiting or refusing food or drink0 (0)2 (4)2 (4)Sleep: Normal8 (15)3 (6)4 (8) More than usual20 (38)21 (40)20 (38) More disturbed than usual9 (17)15 (29)10 (19) A lot more disturbed than usual15 (29)13 (25)18 (35)Recruitment method: Local17 (33)18 (35)10 (19) Remote27 (52)26 (50)31 (60) Community8 (15)8 (15)11 (21)Ethnicity: White47 (90)47 (90)44 (85) Other5 (10)5 (10)8 (15)Diagnosis: Otitis media7 (14)11 (20)8 (14) Respiratory tract infection12 (23)15 (28)17 (33) Non-specific viral illness21 (40)20 (37)16 (31) Other12 (23)8 (15)11 (22)Previous febrile convulsion: Yes2 (4)1 (2)2 (4) No50 (96)51(9)50 (96)Asthma: Yes9 (17)4 (8)6 (12) No43 (83)48 (92)46 (88)*Minimisation criterion (baseline temperature included as continuous variable in all models; baseline discomfort minimised distinguishing the top and bottom two levels because of anticipated frequencies but included as four level categorical variable in all models).The median time between randomisation and giving the first dose of study drug was eight minutes for paracetamol plus ibuprofen and nine minutes for paracetamol and for ibuprofen. The mean number of valid minutes for time without fever (temperature >33°C and <45°C) in the first four hours (240 minutes) was 219 for children receiving paracetamol, 211 for ibuprofen, and 202 for paracetamol plus ibuprofen. The respective times over 24 hours (1440 minutes) were 1078, 1029, and 1051 minutes. For time without fever in the first four valid hours (and the corresponding secondary outcome within 24 valid hours), children receiving paracetamol plus ibuprofen spent more time without fever than those given ibuprofen and, in turn, those given paracetamol (table 2). Fever clearance was faster in children given paracetamol plus ibuprofen than in those given paracetamol but was similar for those given ibuprofen. Children given paracetamol plus ibuprofen spent less time with fever over 24 hours than those given either drug alone. A suggestion was that more fever associated symptoms had normalised in children given ibuprofen than the other treatments at 24 and 48 hours, but by day 5 these trends had largely disappeared.Table 2 Descriptive statistics of outcomes (time without fever and no discomfort) at selected times. Values are numbers (percentages) unless stated otherwiseOutcomesParacetamol (n=52)Ibuprofen (n=52)Paracetamol plus ibuprofen (n=52)Primary outcomesMean (SD) time without fever in first 4 hours (minutes)*116.2 (65.0)156.0 (57.6)171.1 (40.8)No discomfort at 48 hours†34 (65)37 (71)36 (69)Secondary outcomes:Outcomes at 24 hours: Mean (SD) time until first fever clearance (minutes)‡71.0 (69.1)42.2 (33.5)45.5 (34.3) Mean (SD) time without fever in first 24 hours (minutes)*940.3 (362.9)1055.2 (329.7)1217.4 (237.6) No discomfort†22 (44)36 (69)29 (56) Normal activity†20 (40)20 (58)23 (48) Normal appetite†10 (21)14 (27)14 (29) Normal sleep†17 (37)13 (50)20 (37)Outcomes at 48 hours: Mean (SD) temperature (°C)§36.4 (0.89)36.4 (0.85)36.6 (1.01) Normal activity†31 (60)37 (73)28 (54) Normal appetite†21 (41)22 (44)21(41) Normal sleep†27 (52)31 (61)25 (48)Outcomes at day 5: Mean (SD) temperature (°C)**36.2 (0.93)36.1 (0.78)36.0 (0.66) No discomfort†43 (88)38 (81)38 (76) Normal activity†44 (90)39 (85)37 (73) Normal appetite†29 (58)29 (59)32 (62) Normal sleep†31 (62)25 (50)27 (53)*Time spent with temperature less than 37.2°C in first four hours after first dose of drug, using number of valid 30 second interval points from data logger; unknown for zero, one, and two children in three groups, respectively, by four hours, and zero, two, and two, respectively, by 24 hours. Time without fever over first four hours was 48 minutes for paracetamol, 65 minutes for ibuprofen, and 71 minutes for paracetamol plus ibuprofen and for time without fever in first 24 hours was 65 minutes for paracetamol, 73 minutes for ibuprofen, and 84 minutes for paracetamol plus ibuprofen.†Children reported at relevant time to be “normal” (see table 1); denominators may vary owing to missing data (in most cases fewer than four children).‡Time from baseline until temperature first falls below 37.2°C; unknown for five children (zero, two, and three, respectively) and right censored at 240 minutes for three children.§Measured by research nurse; unknown for one, five, and two children, respectively.**Measured by parent; unknown for four, seven, and three children, respectively.Comparative analysesPrimary outcomesStrong evidence was found of more time spent without fever in the first four hours among children given paracetamol plus ibuprofen than those given paracetamol, and likewise for children given ibuprofen than those given paracetamol (table 3). Moreover, both point estimates exceeded the 30 minute target difference, as did the lower confidence limit for the primary comparison. The confidence interval and P value suggest little difference between giving paracetamol plus ibuprofen and giving ibuprofen alone.The low power for fever associated discomfort at 48 hours was reflected by the large P values and wide confidence intervals for all three comparisons, although the largest point estimate and upper confidence limit favoured ibuprofen over paracetamol. The lowest P value from subgroup analyses for the primary outcomes was 0.14.Table 3 Regression models for time without fever over first four hours (240 minutes) and no discomfort at 48 hours, adjusting for minimisationOutcomesPrimary comparisonsSecondary comparison: ibuprofen v paracetamolParacetamol plus ibuprofen v paracetamolParacetamol plus ibuprofen v ibuprofenTime without fever in first 4 hours*: Adjusted difference (minutes)55.316.239.0 95% confidence interval33.1 to 77.5†−7.0 to 39.4†15.9 to 61.0‡ P value<0.001†0.2†<0.001‡No discomfort at 48 hours§: Adjusted odds ratio1.330.891.50 95% confidence interval0.49 to 3.56†0.32 to 2.43†0.53 to 4.26‡ P value0.7†>0.8†>0.5‡*Weighted by number of time points in first four hours contributing valid data on temperature. Positive differences indicate additional minutes below 37.2°C for first named treatment group compared with comparator.†Primary comparisons after applying Dunnett’s correction (approximate P values obtained using extrapolation from limited published values21; uncorrected P values were <0.001 and 0.11 for time without fever, 0.53 and 0.79 for discomfort).‡Secondary comparison after applying Tukey’s correction (P values obtained using interpolation from extensive published values21; <0.001 for temperature, 0.37 for discomfort).§Odds of being well compared with not being well.Secondary outcomesThe comparison of fever clearance was consistent with the primary outcome for time without fever: strong evidence suggested that paracetamol plus ibuprofen had a faster effect than paracetamol alone, and ibuprofen alone had a faster effect than paracetamol alone (table 4). Giving paracetamol plus ibuprofen over 24 hours increased time without fever by 4.4 hours compared with paracetamol and by 2.5 hours compared with ibuprofen.No consistent evidence of effect for fever associated symptoms from 24 hours to day 5 was seen, but odds ratios tended to favour ibuprofen more than the other treatments at 24 and 48 hours (data not shown).Table 4 Regression models for time without fever up to 24 hours, adjusting for minimisationOutcomesPrimary comparisonsSecondary comparison: ibuprofen v paracetamolParacetamol plus ibuprofen v paracetamolParacetamol plus ibuprofen v ibuprofenTime until first fever clearance*: Adjusted difference (minutes)−23.53.0−26.3 95% confidence interval−44.8 to −2.2†−18.3 to 24.4†−48.3 to −4.3‡ P value0.025†>0.8†0.015‡Time without fever in first 24 hours§: Adjusted difference (hours)4.42.51.9 95% confidence interval2.4 to 6.3†0.6 to 4.4†−0.2 to 4.0‡ P value<0.001†0.008†0.076‡*Negative differences indicate that first named treatment group has faster fever clearance time than comparator group.†Primary comparisons after applying Dunnett’s correction (uncorrected P values were 0.016 and 0.75 for fever clearance, <0.001 and 0.005 for time without fever).‡Secondary comparison after applying Tukey’s correction (uncorrected P values were 0.006 for fever clearance, 0.033 for time without fever).§Weighted by number of time points in first 24 hours contributing valid data on temperature; positive differences indicate additional hours with temperature less than 37.2°C for first named treatment group than for comparator.Mean temperature by treatment groupFigure 3 shows the mean temperature every 15 minutes by treatment group with the proportion of children febrile at corresponding two hourly time points. Ibuprofen and paracetamol plus ibuprofen reduced children’s temperatures faster and for longer than paracetamol in the first four hours, and paracetamol plus ibuprofen was superior to either drug alone in reducing mean temperatures over 24 hours. A rise in mean temperature was seen for children in the ibuprofen group, which then fell just after six hours, coinciding with the earliest time that parents were advised that a second dose of ibuprofen could be given. This rise may have been prevented in the other groups by paracetamol, which could have been given at four hours.The mean temperatures in the graph are lower than might be expected biologically. This could be explained by the choice of axillary thermometry, which is known to record temperatures around 0.8°C lower than rectal digital thermometers,23 or by the liberal definition of valid temperature used in this study, or both. A sensitivity analysis excluding temperatures below 35°C raised the mean temperatures but not the relative positions of the group means.Fig 3 Mean temperature over first 24 hours after randomisation, by treatment group. *All children had temperatures greater than 37.2°C at baseline eligibility assessment, as measured by standard digital axillary thermometry. Temperature measured using a data logger was less than 37.2°C for 19 children because of delays between digital thermometry measure and drug dosing and differences between digital and data logger thermometry methodsRelation between discomfort and temperatureGiven the low power for treatment effects on discomfort, a repeated measure analysis was used to explore the relation between all discomfort measures recorded across up to eight time points to 48 hours and their coinciding mean digital axillary thermometer measures. The mean temperatures were 36.4°C for children who scored normal on the discomfort scale, 37.2°C for those who scored not quite normal, 38.1°C for those who scored some pain or distress, and 38.3°C for those who scored crying or very distressed.Adverse effectsThe most common adverse effects were diarrhoea and vomiting, which were equally distributed between groups (table 5). The overall number of children experiencing adverse events was, however, too small to make meaningful comparisons between treatments. Five children were admitted to hospital (constituting serious adverse events21): one child in the paracetamol group, three in the ibuprofen group, and one in the paracetamol plus ibuprofen group. On independent review none was considered to be related to the study process or drugs.Table 5 Five most common adverse effects. Values are numbers of childrenAdverse effectParacetamol (n=52)Ibuprofen (n=52)Paracetamol plus ibuprofen (n=52)Diarrhoea10912Vomiting632Rash221Cough201Cold to touch032Dosing of study drugsAll 52 children in each of the three groups were given, as per protocol, their first dose of study drug under nurse supervision (table 6). The recommended maximum four doses of paracetamol or placebo in the first 24 hours was received by 65% of children given paracetamol, 46% given ibuprofen, and 42% given paracetamol plus ibuprofen, with this recommended maximum exceeded by 12%, 6%, and 8%, respectively. The corresponding percentages receiving the recommended maximum three doses of ibuprofen or placebo in 24 hours were 73%, 75%, and 71% and those exceeding this recommended maximum were 13%, 12%, and 13%. All percentages were much lower at 48 hours.Table 6 Number of doses of paracetamol alone or ibuprofen alone over 24 and 48 hours. Values are numbers (percentages) of childrenDrug use (dose No)Paracetamol (n=52)Ibuprofen (n=52)Paracetamol plus ibuprofen (n=52)Paracetamol or placebo in 24 hours: 152 (100)52 (100)52 (100) 252 (100)49 (94)51 (98) 348 (92)44 (85)47 (90) 434 (65)24 (46)22 (42) 56 (12)3 (6)4 (8)Paracetamol or placebo in 48 hours: 152 (100)52 (100)52 (100) 252 (100)49 (94)51 (98) 350 (96)49 (94)49 (94) 442 (81)39 (75)38 (73) 535 (67)26 (50)24 (46) 620 (38)11 (21)15 (29) 78 (15)6 (12)6 (12) 83 (6)1 (2)1 (2) 9—1 (2)—Ibuprofen or placebo in 24 hours: 152 (100)52 (100)52 (100) 251 (98)48 (92)51 (98) 338 (73)39 (75)37 (71) 47 (13)6 (12)7 (13) 5——2 (4)Ibuprofen or placebo in 48 hours: 152 (100)52 (100)52 (100) 251 (98)49 (94)51 (98) 345 (87)45 (87)46 (88) 432 (62)34 (65)29 (56) 518 (35)5 (10)18 (35) 67 (13)4 (8)10 (19) 7—1 (2)3 (6)BlindingThe success of blinding was assessed at the nurse’s visit at 48 hours, when parents were asked to guess treatment allocation. Taking “I don’t know” responses to either drug as failure to guess correctly, allocation was guessed correctly by 16 (31%) parents in the paracetamol group, 17 (33%) in the ibuprofen group, and 9 (17%) in the paracetamol plus ibuprofen group, compared with the 33% expected by chance. Excluding “I don��t know” responses increased these percentages to 50% (32 parents), 53% (n=32), and 43% (n=21), respectively.DiscussionIn febrile children we found strong evidence of faster time to fever clearance and more prolonged time without fever in the first four hours favouring the use of paracetamol plus ibuprofen and ibuprofen over paracetamol, but no evidence of any difference between paracetamol plus ibuprofen and ibuprofen alone. In the first 24 hours strong evidence suggested more time without fever favouring paracetamol plus ibuprofen over either drug alone. We found no evidence of differences in fever associated discomfort at 48 hours. The frequency of adverse effects did not seem to differ between groups.Comparison with existing literatureUsing continuous thermometry we compared the effects of two antipyretics combined with either drug alone using maximum licensed, repeated doses in children recruited from and managed in the community. Previous studies have recruited from secondary care,11\n12\n14\n15 investigated the effects of single doses,12\n14 and did not use continuous thermometry. The finding that ibuprofen was found to be more effective than paracetamol in the first four hours is consistent with the literature.9Strengths and limitations of the studyThe study has four main strengths. Firstly, its internal validity: randomisation was concealed, nurses and investigators were blinded to allocation, and attrition was minimal. Secondly, the intervention and follow-up periods were long enough to enable a fair comparison between multiple doses of antipyretics with differing times to maximum effect.16 Thirdly, we used continuous thermometry to generate the objective and intuitive outcome of time without fever. Finally, we recruited and followed up children in the community, where most cases of fever are managed.We are aware of five possible weaknesses of the study. Firstly, because we had no placebo only group our data cannot inform the decision on whether to use antipyretics. This was a deliberate design decision as we thought that parents would not have participated if there had been a placebo only group. This judgment is supported by the fact that over 80% of parents in the study said that they would not have participated in such a trial. Three previous trials have, however, shown that paracetamol and ibuprofen given separately are more effective than placebo,6\n7\n8 and one trial found that paracetamol is more effective at relieving fever than unwrapping children.20Secondly, the recruited sample did not give sufficient power to detect plausible differences in discomfort. This is disappointing, given the importance of this question to the public and research community. Other research has, however, suggested that the use of two drugs combined compared with one alone does confer additional benefit on symptoms13 and we did find a relation between increasing discomfort and worsening fever, suggesting that with adequate power the effects on symptoms might have followed those of temperature.Thirdly, an axillary temperature of 37.8°C might not be regarded as denoting fever. Since no agreed definition of fever or how to measure temperature exists,24 to a limited extent its selection was arbitrary. For example, disagreement between thermometer types and measurement sites means this could represent a rectal temperature of as much as 39.7°C.23 Temperature is such a dynamic variable that although many children did not meet our criterion for temperature before randomisation, most were already being treated for a febrile illness and their parents and doctors thought that treatment with up to two drugs was warranted. The mean temperature at baseline was 38.5°C (table 1), a temperature at which 90% of doctors and 70% of nurses would recommend treatment,25 and most of the children were unwell with febrile illness as it affected their comfort, appetite, activity, and sleep.Fourthly, the success of blinding was assessed at the 48 hour nurse visit by asking parents to guess which drugs were active. Overall, the 153 parents who responded were not able to guess treatment, but the 83 who expressed a definite opinion did identify allocation more often than would be expected by chance. Although we carried out blinded taste tests and volunteers could not distinguish placebo from active drugs, some parents may have been better able to do so because they had more time to compare study drugs with known products in the home as well as observing their children’s responses to treatment. Although this could have influenced the parental recording of the discomfort outcome, we do not see how it could influence the outcome of time without fever.Finally, given the challenges of recruitment, our sample might not be representative of the general population. For example, we do not know if the possibility of receiving either or both drugs combined and the severity of the child’s illness influenced parents’ decisions to participate. If this was the case, we think these factors are more likely to be associated with differences in parental attitudes to illness than the children’s response to the drugs. The most common reason for ineligibility was insufficient fever, a factor we think is unlikely to be associated with any other physiological marker of response to drugs.Implications of this researchIt is good practice for parents, nurses, and doctors who have made the decision to treat young, unwell children with fever, to use the minimum number of drugs possible.5 Although other studies have shown that paracetamol is superior to placebo,6\n7\n8 our study suggests that those wanting to achieve faster and more prolonged fever relief in the first four hours should use ibuprofen in preference to paracetamol. Similarly, where symptoms are expected to last at least 24 hours (probably most children with more severe symptoms at the onset of illness), those wanting to maximise the time without fever should probably start with ibuprofen but also consider paracetamol plus ibuprofen. Pragmatically, although our trial design did not specifically address this, we speculate that if a child remains unwell after a first dose of ibuprofen, subsequent alternation of paracetamol and ibuprofen for 24 hours would be more effective than either drug alone. This speculation is supported by a recent study showing that paracetamol was more effective than placebo when added to ibuprofen.14 The decision to start with ibuprofen or paracetamol plus ibuprofen, however, should also be influenced by an assessment of the benefits (an additional 2.5 hours without fever) compared with the risk of unintentionally exceeding the maximum recommended dose owing to the additional complexity of using two drugs. This risk is not theoretical. Even in the context of this supervised trial, between 6% and 13% of parents exceeded the maximum number of recommended doses in the first 24 hours.The pragmatism of the intervention changed with time, moving from efficacy in the first four hours to effectiveness in the second 24 hours. By 48 hours, considerably fewer study drugs were being given and this could partly explain the observed lack of effects on discomfort at this time. In the community, paracetamol and ibuprofen are usually dosed by age, and we recognise that calculating doses by weight means the results may inform primary and secondary care practice more than practice at home. We decided against a dose by age regimen, however, for two reasons. Firstly, given the recommendation of the children’s national service framework to dose by weight26 and the dose by weight presentations in the British national formulary for children,18 we believe that in the future more medicines for children will be given by weight. Secondly, we wanted to ensure that heavier children for their age received a therapeutic dose and to avoid exceeding the normal recommended dose for children who were light for their age. Comparing dose by weight with dose by age shows that children can receive as much as 50% more27 or 50% less paracetamol and 100% more ibuprofen.Medicine bottles in the United States contain dosing advice by both age and weight and although healthcare professionals can clearly calculate dose by weight, we think two steps are needed before parents can routinely use weight to determine dose in other countries. Firstly, studies should investigate the safety implications of any differences between estimates of children’s weights measured by parents using domestic scales (or recently recorded weights in parent held children’s health records) and those measured by professionals using paediatric scales. Secondly, suppliers of antipyretics could consider routinely including dose by weight tables. Given that the complexity of using two drugs over a 24 hour period is more likely to lead to inadvertently exceeding the maximum recommended dose, we also believe that multiple blank charts should be supplied for parents to record when medicines have been given and how much.Recent case reports have highlighted the concern about renal toxicity in dehydrated children given ibuprofen.28\n29 Although this serious effect is rare, we excluded children with dehydration from our trial and believe that ibuprofen should not routinely be given to children with, or at risk of, dehydration. Good evidence shows, however, that ibuprofen is as safe as paracetamol for children with asthma, where there is no evidence of sensitivity to non-steroidal anti-inflammatory drugs.30We agree with the guidelines for fever from the National Institute for Health and Clinical Excellence (NICE) that antipyretics should be used only when children have fever associated with other symptoms,5 although further research is needed to establish the effectiveness of antipyretics for the relief of these symptoms. However, we believe that the guidance on the use of two drugs combined need not be so cautious now that there is good evidence of superiority for both drugs over one drug for increasing time without fever over 24 hours.ConclusionDoctors, nurses, pharmacists, and parents wanting to use medicines to treat young, unwell children with fever should be advised to use ibuprofen first and to consider the relative benefits and risks of using paracetamol plus ibuprofen over a 24 hour period. There is no evidence from the accompanying cost effectiveness evaluation to contradict these findings.17What is already known on this topicParacetamol plus ibuprofen are being increasingly used at home and in primary and secondary care for the relief of fever and its associated symptomsFive previous trials of combined therapy mostly tested single doses for children in secondary care and reached conflicting conclusionsWhat this study addsIn the first four hours, temperature is reduced faster and for longer in children given ibuprofen than in those given paracetamolIn the first 24 hours, children given both drugs spent 4.4 hours less time with fever than those given paracetamol and 2.5 hours less time with fever than those given ibuprofen.Parents and healthcare professionals should consider ibuprofen first and the relative benefits and risks of using combined therapy over 24 hours\n\nREFERENCES:\n1. Hay AD, Heron J, Ness A, the ALSPAC study team. 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Sarrell EM, Wielunsky E, Cohen HA. Antipyretic treatment in young children with fever: acetaminophen, ibuprofen, or both alternating in a randomized, double-blind study. Arch Pediatr Adolesc Med2006;160:197-202.16461878\n14. Nabulsi MM, Tamim H, Mahfoud Z, Itani M, Sabra R, Chamseddine F, et al. Alternating ibuprofen and acetaminophen in the treatment of febrile children: a pilot study. BMC Med2006;4:4.16515705\n15. Kramer LC, Richards PA, Thompson AM, Harper DP, Fairchok MP. Alternating antipyretics: antipyretic efficacy of acetaminophen versus acetaminophen alternated with ibuprofen in children. Clin Pediatr (Phila)2008 Jun 16 [ePub ahead of print].\n16. Kelley MT, Walson PD, Edge JH, Cox S, Mortensen ME. Pharmacokinetics and pharmacodynamics of ibuprofen isomers and acetaminophen in febrile children. Clin Pharmacol Ther1992;52:181-9.1505153\n17. Hollinghurst S, Redmond N, Costelloe C, Montgomery AA, Fletcher M, Peters TJ, et al. Paracetamol plus ibuprofen for the treatment of fever in children (PITCH): economic evaluation of the randomised controlled trial. BMJ2008 (in press).\n18. British Medical Association, Royal Pharmaceutical Society of Great Britain, Royal College of Paediatrics and Child Health. BNF for children. London: BMJ Publishing Group, RPS, RCPCH, 2007.\n19. Treasure T, MacRae KD. Minimisation: the platinum standard for trials? Randomisation doesn’t guarantee similarity of groups; minimisation does [editorial] [see comments]. BMJ1998;317:362-3.9694748\n20. Kinmonth AL, Fulton Y, Campbell MJ. Management of feverish children at home. BMJ1992;305:1134-6.1463951\n22. Zar J. Biostatistical analysis. 2 ed. Englewood Cliffs, NJ: Prentice-Hall, 1994.\n23. Craig JV, Lancaster GA, Williamson PR, Smyth RL. Temperature measured at the axilla compared with rectum in children and young people: systematic review. BMJ2000;320:1174-8.10784539\n24. Michael MS, Kohl KS, Dagan R, Nalin D, Blum M, Jones MC, et al. Fever as an adverse event following immunization: case definition and guidelines of data collection, analysis, and presentation. Vaccine2004;22:551-6.14741143\n25. Sarrell M, Cohen HA, Kahan E. Physicians’, nurses’, and parents’ attitudes to and knowledge about fever in early childhood. Patient Educ Couns2002;46:61-5.11804771\n26. Department of Health. National service framework for children, young people and maternity services. London: Her Majesty’s Stationery Office, 2004.\n27. Bua J, L’Erario I, Barbi E, Marchetti F. When off-label is a good practice: the example of paracetamol and salbutamol. Arch Dis Child2008;93:546-54a.\n28. Mathews John C, Shukla R, Jones CA. Using NSAID in volume depleted children can precipitate acute renal failure. Arch Dis Child2007;92:524-6.17293366\n29. Moghal NE, Hegde S, Eastham KM. Ibuprofen and acute renal failure in a toddler. Arch Dis Child2004;89:276-7.14977711\n30. Lesko SM, Louik C, Vezina RM, Mitchell AA. Asthma morbidity after the short-term use of ibuprofen in children.[see comment]. Pediatrics2002;109:E20.11826230"
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+{
+    "id": "PMC2528935",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528935\nAUTHORS: Reginia H. Y. Yan, Jessica L. Malisch, Robert M. Hannon, Peter L. Hurd, Theodore Garland\n\nABSTRACT:\nThe ratio of the length of the second digit (index finger) divided by the fourth digit (ring finger) tends to be lower in men than in women. This 2D∶4D digit ratio is often used as a proxy for prenatal androgen exposure in studies of human health and behavior. For example, 2D∶4D ratio is lower (i.e. more “masculinized”) in both men and women of greater physical fitness and/or sporting ability. Lab mice have also shown variation in 2D∶4D as a function of uterine environment, and mouse digit ratios seem also to correlate with behavioral traits, including daily activity levels. Selective breeding for increased rates of voluntary exercise (wheel running) in four lines of mice has caused correlated increases in aerobic exercise capacity, circulating corticosterone level, and predatory aggression. Here, we show that this selection regime has also increased 2D∶4D. This apparent “feminization” in mice is opposite to the relationship seen between 2D∶4D and physical fitness in human beings. The present results are difficult to reconcile with the notion that 2D∶4D is an effective proxy for prenatal androgen exposure; instead, it may more accurately reflect effects of glucocorticoids, or other factors that regulate any of many genes.\n\nBODY:\nIntroductionResearchers have long noted that men tend to have lower index∶ring finger ratios than women [1], [2], [3]. Anecdotal reports of subtle differences in such measures of hand shape between mathematicians and engineers, as compared with artists and litterateurs, for example, date back at least a century [4]. This 2D∶4D ratio is lower, more masculine-typical, in both men and women of greater physical fitness, or who participate at more elite levels of athletic competition [5]–[10]. 2D∶4D has been proposed to correlate with health and personality traits because of the common organizational influence of prenatal testosterone across these traits [11]–[14]. Although studies linking prenatal environment to variation in digit ratio do imply a role for fetal testosterone, a direct cause-and-effect relation is far from proven. For example, girls with congenital adrenal hyperplasia have lower, more male-like digit ratios [15], [16]. Congenital adrenal hyperplasia involves disruption of the glucocorticoid synthesis pathway in the adrenal glands and leads to an absence of circulating glucocorticoids, triggering an over-production of androstenedione, which in turn results in abnormally high adrenal androgen production. The ratio of testosterone to estradiol in amniocentesis samples collected in the second trimester correlated with the child's 2D∶4D ratio at two years of age in a sample of 18 boys and 15 girls [17]. Female dizygotic twins having male co-twins have more masculine left hand 2D∶4D than those with female co-twins [18]. Similarly, mice that gestate next to males have larger ratios than those gestating next to females [19], suggesting an effect of fetal testosterone in the opposite direction. Behavioral studies correlating 2D∶4D in humans and mice also suggest similar, but opposite effects: humans with lower 2D∶4D show greater propensity for aggression [20]–[22], whereas inbred mouse strains with larger 2D∶4D were more likely to bite while being handled [23]. Lower digit ratios are associated with greater physical fitness or athletic competitiveness in men and women [5]–[10], but mouse strains with higher digit ratios tend (p = 0.058) to show higher daily activity levels (measured by the total number of beam breaks per day in an individual live-in cage) [23].The overwhelming trend in human digit ratio studies shows more masculinized 2D∶4D to be associated with more masculinized behavior within each sex, so the studies linking sporting achievement and digit ratio make intuitive sense. However, the idea that 2D∶4D simply reflects the organizational effects of testosterone cannot be the whole story. The emphasis on prenatal testosterone as the major determinant of digit ratio runs counter to the fact that variation in digit ratio among human ethnic groups is far larger than differences between the sexes [24]–[26]. Similarly, lab mice show large inter-strain differences in 2D∶4D [23], but evidence for a sex difference in lab mice is mixed: two small studies show a higher 2D∶4D in females [27], 28, while a larger study finds no difference [23]. Whether digit ratio in mice is sexually dimorphic, and whether variation in rodent digit ratio can serve as a reliable proxy for prenatal environment in behavioral studies, is a matter of debate [29], [30]. Some [e.g. 11] have argued that the digit ratio effect is driven by regulation of HOX genes by circulating androgens. Evidence of digit ratio effects from other taxa have been ambiguous. In lizards, sex differences have been reported in opposing directions for different species within the same study [31]. Studies of Anolis carolinensis have reported significant effects of sex as well as significant differences between laboratories/populations [32], but another smaller study reports no significant difference [33]. Similar patterns of results have been reported in birds, where sex differences found in zebra finches [34] were not replicated by a larger study [35], and in pheasants where sex differences in 2D∶4D were found in one population [36], but not another [37]. Given the highly conserved nature of HOX genes, and their pleiotropic effect on morphological development of both gonads and fingers, one ought to expect a highly robust sex effect on digit ratio across taxa [28].We used an experimental evolution approach [38] to investigate the link between behavior and digit ratios in laboratory house mice. We compared mice from lines that have been selectively bred for high rates of voluntary exercise with their unselected control lines. Mice from the selected (High Runner, HR) lines exhibit many differences relative to control lines, including higher voluntary wheel-running speeds, home-cage activity, maximal aerobic capacity, and predatory aggression, but no difference in maternal or intermale aggression [39]–[42]. Importantly, the HR lines also show 2-fold elevated baseline circulating corticosterone, but no apparent difference in circulating testosterone [40], [42], [43]. We hypothesized that, if digit ratio reflects the organizational effects of testosterone, then we would find a consistent sex difference in the mice. The digit-ratio-reflects-testosterone hypothesis predicts no difference between HR and control lines, given the absence of an effect of the selective-breeding regime on testosterone levels. We found a sex difference, higher 2D∶4D in females than males, statistically significant in the selected lines only, and an effect of selective breeding, higher 2D∶4D ratios in HR lines, in both sexes. The latter effect seems to contradict human studies, where masculinized digit ratios are associated with greater physical fitness or sporting achievement.ResultsWe compared hind limb 2D∶4D, measured from the mid-point of the basal crease to digit tip (Fig. 1), of the four HR lines (male∶female N = 66∶79, 74∶89, 69∶62, 77∶69) with those of the four Control (C) lines (N = 48∶61, 49∶54, 58∶58, 53∶47).10.1371/journal.pone.0003216.g001Figure 1Measurement of digit lengths.Right rear palm of male mouse from a line bred for high running, illustrating digit lengths, measured from the mid-point of the basal crease to digit tip. The 2D∶4D ratio was 573.6∶585.0 pixels, yielding a 2D∶4D of 0.9805.Overall, 2D∶4D was higher in females than males, and in HR lines than Control lines (Fig. 2). On the right limb, differences were significant for sex (F(1,6) = 12.88, p = 0.0115) and linetype (F(1,6) = 11.76, p = 0.0140), without significant interaction between these main effects (F(1,6) = 2.99, p = 0.1345) (for the random effects, χ2 = 5.10, d.f. = 3, P = 0.0779). Results were similar on the left limb, but did not reach statistical significance (sex F(1,6) = 4.88, p = 0.0691; linetype F(1,6) = 4.71, p = 0.0730; interaction F(1,6) = 0.19, p = 0.6803) (for the random effects, χ2 = 37.11, d.f. = 3, P<0.0001). The finding of significant effects only on the right side fits the general pattern of previous research on human beings [11], [12].10.1371/journal.pone.0003216.g002Figure 2Digit ratios by sex and selection line.2D∶4D finger length ratios by sex and selection regime for the eight lines of mice. Control are lines 1, 2, 4 and 5 (lab designations), High Runner selected lines are 3, 6, 7 and 8), open bars indicate females, and shaded bars males. Values are least square means and associated standard errors from models (separately for Control and Selected lines) including sex, line, sex-by-line interaction, and generation as fixed effects (SAS Procedure Mixed: family nested within line and the sex-by-family(line) interaction were random effects). See text for P values and for results of analyses comparing the four Control with the four High Runner lines.Although the interaction between sex and linetype was not statistically significant, Figure 2 suggests that the significant sex effect on the right paw is driven by the HR lines. Indeed, separate analyses of the Control and HR lines showed no sex difference for either limb in Control lines (right F(1,91) = 1.63, p = 0.2047; left F(1,91) = 1.76, p = 0.1879), whereas HR lines showed a sex difference in 2D∶4D on both rear limbs (right F(1,81) = 14.56, p = 0.0003; left F(1,81) = 5.74, p = 0.0189).DiscussionSelective breeding for the behavioral trait of high voluntary exercise (wheel running) raised the 2D∶4D digit ratio in these mice, suggesting that the selection regime “feminized” them, as females in this study had larger 2D∶4D ratios than males. The direction of the sex effect, larger in females than in males, agrees with those studies that have found sex differences in mice [27], [28]. Similarly, the effect of selective breeding parallels the trend among inbred strains where higher total daily activity correlates positively with digit ratio [23]. Therefore, the present results seem to contradict the human studies, where masculinized digit ratios are associated with greater physical fitness or sporting achievement.Several possible explanations exist for this discrepancy, the first being that the HR lines are indeed the more feminized, but that high voluntary wheel running in mice is not a homologous trait to the traits assayed in the human studies. Female mice run more than males [42]. In human studies, the physical fitness measures used can be broadly classified as either level of competition in hierarchically organized sports [5]–[7] or objective performance scores on athletic tasks [8]–[10]. In a study of an otherwise unselected sample of 77 women and 102 men (mostly university students) [8], the number of hours of exercise a week was measured, as a potentially confounding variable, and correlated significantly, and positively, with digit ratio (females, left hand: r = 0.27, P = 0.017; males, right hand: r = 0.38, P = 0.0001; note that these sex-by-hand combinations were the only ones correlating with physical fitness in the original study). This suggests that voluntary exercise in humans, unlike physical fitness, is associated with less masculinized 2D∶4D. Here, we found no such relationship in mice. In any case, the literature linking masculinized 2D∶4D with greater physical fitness in not unequivocal; for example, lower 2D∶4D is associated with earlier age of first myocardial infarction in men [28].A second possible explanation is that higher exercise rates are in fact more “masculinized,” but that lower digit ratios do not indicate greater masculinization in these mice. The present data do not provide unequivocal support for larger 2D∶4D in females. The unselected control lines, like those in [23], do not show statistically significant sexual dimorphism. Other evidence, such as intrauterine position [19], suggests an effect in the opposite direction, higher in more masculinized individuals. On balance, the lack of any sex effect in that direction suggests strongly that if digit ratio indicates masculinization, then the high-running lines are the more feminized, and the possibility that lower digit ratios are more masculinized is a poor candidate explanation for our results.A third possible explanation for our results is that propensity for voluntary physical exercise and 2D∶4D are not linked to masculinization or, if they are, not through prenatal androgen exposure. Although 2D∶4D was higher in high-running lines of mice, they do not have higher testosterone, at least as adults [40]. However, the sex and selection regime effects on 2D∶4D match those for adult baseline circulating corticosterone: females have higher corticosterone levels than males, and HR lines have higher levels than Controls [42], [43]. Similarly, females have higher 2D∶4D than males, and HR have higher 2D∶4D than Controls. Although increased circulating glucocorticoids are generally thought to depress testosterone, some evidence suggests that they are positively correlated in the human fetus [44]. We are unaware of any published work directly examining the effects of glucocorticoids on digit ratio. However, experimental manipulations of hormones other than testosterone have been demonstrated to influence digit ratio. Male, but not female, ring necked-pheasants show decreased right limb 2D∶4D in response to increased egg estradiol [36], whereas they show an increase in left limb 2D∶3D–not 2D∶4D–in females, but not males, in response to increased egg testosterone [37]. Given the many factors that have the ability to affect digit ratio, it is clearly more complicated than a simple testosterone-driven manliness metric. The significant additive genetic effects on human digit ratios imply a possible common effect of genes on both 2D∶4D and behavioral traits, aside from any common hormonal influence [44], [45]. Our results cast some doubt on 2D∶4D serving as an indicator of prenatal testosterone exposure, per se. Nonetheless, our results support the idea that relative digit length reflects some endocrinology-related aspect of development. Further investigation of the interacting effects of genetic variation, perinatal hormone levels, and such environmental influences as prenatal maternal stress and perinatal maternal care will be required before the full story is understood.Materials and MethodsThe selective breeding methodology is described in [39]. To achieve a large sample size (total N = 1,013), mice were sampled from generations 45–48. Digits were measured by RHY (blind to generation, sex, line, and linetype), following the same procedures as in our previous work [13], [14]. Briefly, hind paws preserved in 4% paraformaldehyde were straightened and fixed palm-up to an adhesive backing, then photographed via stereoscope. Digit length was measured from the mid-point of the basal crease to digit tip (Fig. 1). Each paw was photographed and measured twice while the paw was left in the same position. The mean value of the two measures (which were highly correlated, Pearson's r = 0.9 for both left and right paws), was used for analysis. To calculate the reliability of the 2D∶4D measures, we re-imaged a random sample of 50 mice several months after the original images were taken, then calculated the intraclass correlation coefficient following [47]. Left paws: ICC = 0.66 (95%CI: 0.472<ICC<0.792), F(49,50) = 4.89, P≪0.001), Right paws: ICC = 0.544 (95%CI: 0.317<ICC<0.713), F(49,50) = 3.39, P≪0.001).We used REML estimation in SAS Procedure Mixed (SAS Institute, Cary, NC) to perform mixed-model ANOVA. Main fixed effects were linetype (HR vs. C), sex, and the sex-by-linetype interaction (generation was included as a nuisance variable). Random effects were family nested within line, replicate lines nested within linetype, and the sex-by-line(linetype) interaction term (the number of families in each of the eight lines was 32, 39, 27, 34, 28, 33, 28 & 28 for lines 1 through 8 respectively; note lines designated 1, 2, 4 & 5 were control lines, while 3, 6, 7 & 8 were selected lines). We also separated HR and C lines for two-way ANOVAs of sex and line, both treated as fixed, with family as random nested within line (see Fig. 2). Two-tailed P values are presented.\n\nREFERENCES:\n1. GeorgeR\n1930\nHuman finger types.\nAnat Rec\n46\n199\n204\n2. KloepferHW\n1946\nAn investigation of 171 possible linkage relationships in man.\nAnn Eugen\n13\n35\n71\n20982098\n3. PhelpsVR\n1952\nRelative index finger length as a sex-influenced trait in man.\nAm J Hum Genet\n4\n72\n89\n14943709\n4. BakerF\n1888\nAnthropological notes on the human hand.\nAm Anthropol\n1\n51\n76\n5. ManningJTTaylorRP\n2001\nSecond to fourth digit ratio and male ability in sport: implications for sexual selection in humans.\nEvol Hum Behav\n22\n61\n69\n11182575\n6. PokrywkaLRachońDSuchecka-RachońKBitelL\n2005\nThe second to fourth digit ratio in elite and non-elite female athletes.\nAm J Hum Biol\n17\n796\n800\n16254897\n7. PaulSNKatoBSHunkinJLVivekanandanSSpectorTD\n2006\nThe Big Finger: the second to fourth digit ratio is a predictor of sporting ability in women.\nBr J Sports Med\n40\n981\n983\n17008344\n8. HönekoppJManningJTMüllerC\n2006\nDigit ratio (2D∶4D) and physical fitness in males and females: evidence for effects of prenatal androgens on sexually selected traits.\nHorm Behav\n49\n545\n549\n16403410\n9. ManningJT\n2002\nThe ratio of 2nd to 4th digit length and performance in skiing.\nJ Sports Med Phys Fitness\n42\n446\n450\n12391439\n10. ManningJTMorrisLCaswellN\n2007\nEndurance running and digit ratio (2D∶4D): Implications for fetal testosterone effects on running speed and vascular health.\nAm J Hum Biol\n19\n416\n421\n17420996\n11. ManningJT\n2002\nDigit ratio: a pointer to fertility behavior and health\nNew Brunswick, NJ\nRutgers University Press\n12. PutzDAGaulinSJCSporterRJMcBurneyDH\n2004\nSex hormones and finger length What does 2D∶4D indicate?\nEvol Hum Behav\n25\n182\n199\n13. McIntyreMH\n2006\nThe use of digit ratios as markers for perinatal androgen action.\nReprod Biol Endocrinol\n4\n10\n16504142\n14. WilsonGD\n1983\nFinger-length as an index of assertiveness in women.\nPers Individ Diff\n4\n111\n112\n15. ÖktenAKalyoncuMYarişN\n2002\nThe ratio of second- and fourth-digit lengths and congenital adrenal hyperplasia due to 21-hydroxylase deficiency.\nEarly Hum Devel\n70\n47\n54\n12441204\n16. BrownWMHinesMFaneBABreedloveSM\n2002\nMasculinized finger length patterns in human males and females with congenital adrenal hyperplasia.\nHorm Behav\n42\n380\n386\n12488105\n17. LutchmayaSBaron-CohenSRaggattPKnickmeyerRManningJT\n2004\n2nd to 4th digit ratios fetal testosterone and estradiol.\nEarly Hum Devel\n77\n23\n28\n15113628\n18. van AndersSMVernonPAWilburCJ\n2006\nFinger-length ratios show evidence of prenatal hormone-transfer between opposite-sex twins.\nHorm Behav\n49\n315\n319\n16143332\n19. HurdPLBaileyAACongalPYanRHGreerJJPagliardiniS\n2008\nIntrauterine position effects on anogenital distance and digit ratio in male and female mice.\nArch Sex Behav\n37\n9\n18\n18080736\n20. BenderliogluZNelsonRJ\n2004\nDigit length ratios predict reactive aggression in women but not in men.\nHorm Behav\n46\n558\n564\n15555497\n21. BaileyAAHurdPL\n2005\nFinger length ratio predicts physical aggression in men but not women.\nBiol Psych\n68\n215\n222\n22. McIntyreMHBarrettESMcDermottRJohnsonDDPCowdenJ\n2007\nFinger length ratio (2D∶4D) and sex differences in aggression during a simulated war game.\nPers Individ Diff\n42\n755\n764\n23. BaileyAAWahlstenDHurdPL\n2005\nDigit ratio (2D∶4D) and behavioral differences between inbred mouse strains.\nGenes Brain Behav\n4\n318\n323\n16011578\n24. ManningJTHenziPVenkatramanaPMartinSSinghD\n2003\nSecond to fourth digit ratio: ethnic differences and family size in English Indian and South African populations.\nAnn Hum Biol\n30\n579\n588\n12959899\n25. ManningJTBarleyLWaltonJLewis-JonesDITriversRLSinghD\n2000\nThe 2nd∶4th digit ratio sexual dimorphism population differences and reproductive success: evidence for sexually antagonistic genes?\nEvol Human Behav\n21\n163\n183\n26. LoehlinJCMcFaddenDMedlandSEMartinNG\n2006\nPopulation differences in finger-length ratios: Ethnicity or latitude?\nArch Sex Behav\n35\n739\n742\n16688484\n27. BrownWMFinnCJBreedloveSM\n2002\nSexual dimorphism in digit-length ratios of laboratory mice.\nAnat Rec\n267\n231\n234\n12115273\n28. ManningJTCallowMBundredPE\n2003\nFinger and toe ratios in humans and mice: Implications for the aetiology of diseases influenced by HOX genes.\nMed Hypotheses\n60\n340\n343\n12581609\n29. VoracekM\n2006\nOf mice and men - cross-species digit ratio (2D∶4D) research: comment on Bailey Wahlsten and Hurd (2005).\nGenes Brain Behav\n5\n299\n300\n16594983\n30. HurdPLWahlstenD\n2006\nNo sex difference in mouse digit ratio: reply to Voracek.\nGenes Brain Behav\n5\n300\n302\n31. RuboliniDPupinFSacchiRGentilliAZuffiMAL\n2006\nSexual dimorphism in digit length ratios in two lizard species.\nAnat Rec\n288A\n491\n497\n32. ChangJLDoughtySWadeJLovernMB\n2006\nSexual dimorphism in the second-to-fourth digit length ratio in green anoles, Anoles carolinensis (Squamata: Polychrotidae), from the south-eastern United States.\nCan J Zool\n84\n1489\n1494\n33. LombardoMPThorpePA\n2008\nDigit ratios in green Anolis lizards (Anolis carolinensis).\nAnat Rec\n291\n433\n440\n34. BurleyNTFosterVS\n2004\nDigit ratio varies with sex, egg order, and strength of mate preference in zebra finches.\nProc R Soc Lond B\n271\n239\n244\n35. ForstmeierW\n2005\nQuantitative genetics and behavioural correlates of digit ratio in the zebra finch.\nProc R Soc Lond B\n272\n2641\n2649\n36. SainoNRuboliniDRomanoMBoncoraglioG\n2007\nIncreased egg estradiol concentration feminizes digit ratios of male pheasants (Phasianus colchicus).\nNaturwissenschaften\n94\n207\n212\n17136513\n37. RomanoMRuboliniDMartinelliRAlquatiABSainoN\n2005\nExperimental manipulation of yolk testosterone affects digit length ratios in the ring-necked pheasant (Phasianus colchicus).\nHorm Behav\n48\n342\n346\n15878573\n38. GarlandTJrRoseMR\n2009\nExperimental evolution: concepts, methods, and applications of selection experiments\nBerkeley CA\nUniversity of California Press\nIn press\n39. SwallowJGCarterPAGarlandTJr\n1998\nArtificial selection for increased wheel-running behavior in house mice.\nBehav Genet\n28\n227\n237\n9670598\n40. GammieSCHasenNSRhodesJSGirardIGarlandTJr\n2003\nPredatory aggression but not maternal or intermale aggression is associated with high voluntary wheel-running behavior in mice.\nHorm Behav\n44\n209\n221\n14609543\n41. RezendeELGomesFRMalischJLChappellMAGarlandTJr\n2006\nMaximal oxygen consumption in relation to subordinate traits in lines of house mice selectively bred for high voluntary wheel running.\nJ Appl Physiol\n101\n477\n485\n16601309\n42. MalischJL, CWGomesFRChappellMAGarlandTJr\n2008\nCircadian pattern of total and free corticosterone concentrations corticosteroid-binding globulin and physical activity in mice selectively bred for high voluntary wheel-running behavior.\nGen Comp Endocrinol\n156\n210\n217\n18329645\n43. MalsichJLSaltzmanWGomesFRRezendeELJeskeDR\n2007\nBaseline and stress-induced plasma corticosterone concentrations of mice selectively bred for high voluntary wheel running.\nPhys Biochem Zool\n80\n146\n156\n44. GitauRAdamsDFiskNMGloverV\n2005\nFetal plasma testosterone correlates positively with cortisol.\nArch Dis Child Fetal Neonatal Ed\n90\nF166\n169\n15724043\n45. PaulSNKatoBSCherkasLFAndrewTSpectorTD\n2006\nHeritability of the second to fourth digit ratio (2D∶4D): A twin study.\nTwin Res Hum Genet\n9\n215\n219\n16611491\n46. GobroggeKLBreedloveSMKlumpKL\n2008\nGenetic and environmental influences on 2D∶4D finger length ratios: A study of monozygotic and dizygotic male and female twins.\nArch Sex Behav\n37\n112\n118\n18074216\n47. SokalRRRohlfFJ\n1995\nBiometry, 3rd ed\nNew York\nWH Freeman"
+}

batch_10/PMC2529272.json

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+{
+    "id": "PMC2529272",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529272\nAUTHORS: Andrew L Snow, João B Oliveira, Lixin Zheng, Janet K Dale, Thomas A Fleisher, Michael J Lenardo\n\nABSTRACT:\nBackgroundUpon repeated or chronic antigen stimulation, activated T cells undergo a T cell receptor (TCR)-triggered propriocidal cell death important for governing the intensity of immune responses. This is thought to be chiefly mediated by an extrinsic signal through the Fas-FasL pathway. However, we observed that TCR restimulation still potently induced apoptosis when this interaction was blocked, or genetically impaired in T cells derived from autoimmune lymphoproliferative syndrome (ALPS) patients, prompting us to examine Fas-independent, intrinsic signals.ResultsUpon TCR restimulation, we specifically noted a marked increase in the expression of BIM, a pro-apoptotic Bcl-2 family protein known to mediate lymphocyte apoptosis induced by cytokine withdrawal. In fact, T cells from an ALPS type IV patient in which BIM expression is suppressed were more resistant to restimulation-induced death. Strikingly, knockdown of BIM expression rescued normal T cells from TCR-induced death to as great an extent as Fas disruption.ConclusionOur data implicates BIM as a critical mediator of apoptosis induced by restimulation as well as growth cytokine withdrawal. These findings suggest an important role for BIM in eliminating activated T cells even when IL-2 is abundant, working in conjunction with Fas to eliminate chronically stimulated T cells and maintain immune homeostasis.ReviewersThis article was reviewed by Dr. Wendy Davidson (nominated by Dr. David Scott), Dr. Mark Williams (nominated by Dr. Neil Greenspan), and Dr. Laurence C. Eisenlohr.\n\nBODY:\nBackgroundProper homeostasis is achieved during an immune response by controlling the appropriate size and activity of the effector T cell pool to maximize immunity and minimize immunopathology. After an immune response, homeostasis depends on the efficient contraction of the expanded T effector pool. Both processes require the selective death of effector T cells [1-5]. When resting T cells become activated and proliferate under the influence of growth cytokines, they display heightened sensitivity to apoptosis [3,5,6]. The mechanisms by which apoptosis is provoked have been thought to differ depending on the level of antigen in the T cell milieu. In one simple schema, T cell apoptosis proceeds through either an \"intrinsic\" (Bcl-2 superfamily/mitochondrial-dependent) program when antigen levels are low, or an \"extrinsic\" (Fas/CD95/APO-1/death receptor-mediated) pathway under conditions of high or repeated antigen stimulation [1,4,5,7]. In the first case, antigen clearance at the conclusion of an immune response results in diminished growth and survival cytokines (such as interleukin-2 (IL-2)), thus activating the mitochondrial death program. Cytokine withdrawal apoptosis (CWA) dramatically reduces the expanded T effector population to re-establish homeostasis, but permits a small population to persist as memory T cells. CWA is principally regulated by the pro- and anti-apoptotic members of the Bcl-2 family. In particular, the pro-apoptotic \"BH3-only\" proteins Bim and Puma have been implicated in CWA, as revealed by expanded memory T cells in knockout mice [8-10]. These \"BH3 only\" members of the Bcl-2 superfamily cause caspase activation and apoptosis by binding pro-survival congeners and releasing the proapoptotic proteins Bax and Bak [11]. Moreover, we recently discovered that a gain-of-function mutation in N-RAS, which suppresses Bim expression via constitutive extracellular signal-related kinase (ERK) activation, could cause a novel form of ALPS in humans [12]. Indeed, Bim expression is tightly controlled by several transcriptional and post-translational mechanisms that underscore its role in central and peripheral T cell tolerance [13].On the other hand, the extrinsic apoptosis pathway involves restimulation of activated T cells with high doses of antigen during the immune response; a pathway often referred to as \"activation-induced cell death (AICD)\" [1,7,14]. However, it is important not to obfuscate the critical functional distinction between \"activation\" – the process entrained to the antigen receptor that causes resting cells to cycle, expand, and acquire effector function – and the death mechanism induced by TCR restimulation of those effector T cells that counterposes their expansion. So the term \"TCR restimulation\" or \"TCR-induced\" apoptosis will be used herein. The key immunoregulatory consideration is why restimulation by same antigen that produced the immune response, can kill the participating T cells in a highly specific way. At first glance, this event would seem to debilitate the immune response since the antigen, and presumably its pathogenic source, are still present. However, it is best understood as a negative feedback mechanism that constrains effector T cell proliferation to avoid immunopathology, previously termed \"propriocidal\" regulation [3,4,6]. Propriocidal or TCR-induced death increases proportionately with high or persistent levels of antigen in IL-2. TCR-induced death has hitherto been primarily equated with the Fas death receptor. Indeed, the upregulation of Fas ligand (FasL) on the surface of restimulated T cells engages Fas on effector T cells in cis (\"suicide\") or in trans (\"fratricide\") leading to apoptosis [15-18]. Moreover, debilitating mutations in Fas or FasL result in defective lymphocyte homeostasis and autoimmunity first characterized in mice (lpr and gld, respectively) and later in humans with ALPS type Ia or Ib [19,20].The Bim vs. Fas paradigm recently restated for intrinsic vs. extrinsic T cell apoptosis is appealing in its simplicity but illusory. For instance, other BH3-only proteins such as PUMA are likely instrumental in CWA [9,10]. Also, the evidence suggests that Fas may not be the sole mediator of TCR-induced death and that TNF or nonapoptotic pathways may be involved [21,22]. Data from conditional knockout mice in which Fas is ablated or blocked in distinct hematopoietic compartments indicate that Fas-mediated apoptosis may also counter autoimmunity by ensuring the removal of antigen presenting cells, including B cells and dendritic cells rather than T cells [23,24]. Although autoreactive T cells accumulate in T cell-specific Fas knockout mice, surprisingly, loss of Fas confers no selective survival advantage for T cells exposed to repeated antigen challenge [24]. Also, Fas engagement can intersect with the intrinsic pathway through a caspase-8 activating cleavage of Bid – a Bcl-2 superfamily member that can trigger mitochondrial apoptosis. Based on these insights, we asked whether death effector pathways other than Fas, including intrinsic signals routed through mitochondrial activation, were important for TCR-induced death of human T cells.In re-examining human T cells in which FAS signaling is blocked or genetically impaired, we found that TCR-induced apoptosis can proceed through rapid induction of BIM expression in the absence of FAS signals, which contributes to mitochondrial permeabilization and cell death in the presence of IL-2. Knockdown of BIM expression partially rescued cells from TCR-induced death, particularly for CD8+ human T cells. Moreover, we show that TCR-induced apoptosis is normal for ALPS Ia patients displaying elevated BIM expression, but impaired in an ALPS type IV patient in which BIM expression is repressed. Collectively, these data indicate that FAS and BIM can cooperate as independent effector molecules in TCR-induced apoptosis. Our results show BIM plays a key role in T cell contraction even when cytokines are abundant, indicating that FAS- and BIM-mediated T cell apoptosis are not mutually exclusive pathways as recently reinforced in the literature [7].MethodsCells and TreatmentsPatients were enrolled and blood samples were obtained with informed consent under protocols approved by the National Institutes of Health (NIH). Peripheral blood lymphocytes (PBL) from normal donors were isolated by Ficoll density gradient centrifugation, and T cells were activated by either 5 μg/ml ConA or 1 μg/ml OKT3 mAb (Ortho Biotech, Raritan, NJ) plus 25 U/ml rhIL-2 (Peprotech, Rocky Hill, NJ), washed 3× in PBS, then cultured in 100 U/ml rhIL-2 for at least 7 days before apoptosis assays were performed. Activated T cell subsets were separated using CD4 or CD8 Microbeads and MACS magnetic bead cell separation (Miltenyi Biotec, Auburn, CA). In some experiments, inhibitors to caspase 8 (IETD-fmk) or caspase 9 (LEHD) (BioVision, Palo Alto, CA) were added at 20 μM. Caspase 9 enzymatic activity was measured using a Caspase 9 Colorimetric Assay Kit (BioVision) according to the manufacturer's instructions.Flow CytometryApoptosis assays were performed as previously described [12]. Briefly, activated T cells were resuspended in fresh media + IL-2 and stimulated for 24 h with soluble OKT3 mAb, agonistic anti-Fas mAb APO1.3 (Alexis, San Diego, CA) plus 200 ng/ml Protein A, or 2 μM staurosporine. In some experiments, 1 μg/ml of an antagonistic Fas blocking Ab (clone SM1/23, Alexis) was added to cells 30 minutes prior to OKT3 restimulation. The level of apoptosis was determined by staining with 1 μg/ml propidium iodide and flow cytometry analysis using constant time acquisition as previously described. Mitochondrial permeability was measured by staining with 40 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6) (EMD Biosciences, San Diego, CA) for 15 min at 37°C before flow cytometry analysis. For surface staining, cells were stained with 5 μg anti-CD4-fluorescin isothiocyanate (FITC), anti-CD8-phycoerythrin (PE), or anti-CD95-PE (BD Biosciences).Electron MicroscopyTreated cells (5 × 106) were pelleted and overlaid with 2% glutaraldehyde in 0.1 M cacodylate buffer fixative for 2 h at room temperature (RT). Sample preparation and electron microscopy was performed at the Image Analysis Laboratory of the National Cancer Institute (Frederick, MD).Mircoarray AnalysisRNA was isolated from two normal donor activated T cells at 0 or 6 h after OKT3 restimulation using Trizol (Invitrogen) and RNeasy mini-columns (Qiagen, Valencia, CA). Purified RNA was amplified using the Ovation Aminoallyl Amplification System (NuGEN, San Carlos, CA), labeled with Cy5 using the Cy5 Reactive Dye Pack (GE Healthcare, Piscataway, NJ), and cleaned up using Vivaspin columns (VivaScience AG, Hanover, Germany). Amplified RNA (2 μg) was hybridized to Hsbb 23K human spotted arrays (NIAID Mircoarray Research Facility) versus Cy3-labeled reference RNA pooled from six normal donor cycling T cells. Data was analyzed using GenePix and mAdb software.ImmunoblottingCells were lysed in 1% NP-40 lysis buffer for 15 min on ice, then cleared by centrifugation. Protein concentration was determined by BCA assay (Pierce, Rockford, IL), and 20–30 μg total protein was separated by SDS-PAGE. Blots were probed with the following antibodies (Abs): anti-BIM (Stressgen, Ann Arbor, MI); anti-BAX, anti-cytochrome c (clone 7H8.2C12), anti-BCL-xL, anti-BCL-2, anti-MCL-1 (BD Pharmingen); anti-PUMA (Alexis); anti-β-actin (clone AC-15, Sigma). Bound Abs were detected using appropriate horseradish peroxidase-conjugated secondary Abs (Southern Biotech, Birmingham, AL) and ECL (Pierce).siRNA TransfectionsActivated human PBL were transfected with 200 pmol of either specific small interfering RNA oligoribonucleotides (siRNA) or a non-specific (NS) control oligo (Invitrogen, Carlsbad, CA) using the Amaxa Nucleofection system (Amaxa, Koln, Germany). Assessment of knockdown efficiency and all subsequent assays were performed 4 days (human) post-transfection. siRNA sequences are available from Invitrogen (Stealth Select).Results and discussionTCR restimulation induces apoptosis signals independent of FASTo examine TCR-induced death in human T cells, activated peripheral blood lymphocytes (PBL) from normal donors were restimulated with the anti-CD3 mAb OKT3 after cycling in IL-2 for 7–14 days. The majority of these cells are CD4+ and CD8+ T cells, with the latter generally more abundant in culture. Data was obtained for numerous human donors. We found that apoptosis was readily induced in restimulated T cells, marked by chromatin condensation and shrinkage (Figure 1A). This was followed by loss of membrane integrity due to secondary necrosis. Apoptosis was verified by PI exclusion; however, we noted that blocking FAS with an antagonistic Ab (SM1/23) provided only partial protection against TCR-induced death (Figure 1B). Flow cytometric analysis of restimulated T cells also confirmed cell shrinkage and loss of mitochondrial membrane potential, as indicated by decreased DiOC6 staining following 12 h of OKT3 treatment, signifying apoptosis (Figure 1C). Again, blocking Fas with an antagonistic Ab (SM1/23) only partially rescued this drop in mitochondrial membrane potential and cell viability. Remarkably, T cells from an ALPS Ia patient with a FAS death domain mutation also showed only a modest loss of mitochondrial membrane potential and viability (Figure 1C), suggesting a mitochondria-dependent apoptotic signal could proceed despite compromised FAS function. Similarly, cytochrome c released from mitochondria in response to OKT3 restimulation was only modestly decreased by FAS blockade (Figure 1D). We also tested caspase 9 activation, which occurs downstream of cytochrome c release and \"apoptosome\" formation. As expected, caspase 9 activation was only partially reduced in restimulated cells in the presence of FAS blocking Ab, but completely abrogated in the presence of the caspase 9 specific inhibitor LEHD-fmk (Figure 1E). In contrast, the SM1/23 Ab effectively blocked APO1.3 anti-Fas induced apoptosis, indicating that the cells were competent for FAS-mediated death (Additional File 1). Taken together, our data confirms that TCR-induced death relies in part on intrinsic mitochondrial signals triggered independently of FAS-FASL interactions.Figure 1TCR re-stimulation signals mitochondrial-dependent apoptosis independent of FAS. (A) Electron micrographs (upper panels, 2500× magnification or lower panels, 10000×) of activated human PBL either not restimulated (NRS) or restimulated with OKT3 mAb for 18 h. Arrows indicate apoptotic cells. (B) Activated human T cells were untreated (NRS) or restimulated with OKT3 for 24 h in the presence of FAS blocking Ab (SM1/23) or isotype control Ab. Cells were stained with PI and analyzed by flow cytometry; gates indicate % viable cells. (C) Activated human T cells from a normal donor or ALPS 1a patient were untreated (NRS) or restimulated with OKT3 for 12 h in the presence of FAS blocking Ab (SM1/23) or isotype control Ab. Cells were stained with DiOC6 and analyzed by flow cytometry (right panels). Viable gates are shown at left, and the percentage of DiOC6 low cells are indicated in the histograms on the right. (D) Cytosolic extracts from activated human PBL were immunoblotted for the presence of cytochrome c following stimulation with OKT3 or staurosporine (STS) for the indicated timepoints, in the presence or absence of SM1/23 Ab. (E) Lysates prepared as described in (D) were incubated with the caspase 9 specific substrate LEHD-pNA for 2 h, and caspase 9 enzymatic activity was quantitated as OD at 405 nm minus background (OD405 at 5 min).Role for BIM induction in TCR-induced deathInitial studies of AICD indicated that de novo transcription was required for the execution of apoptosis in response to T cell restimulation[17]. Since our data pointed toward a mitochondrial component, we surveyed expression of several pro- and anti-apoptotic BCL-2 family members using microarrays following TCR restimulation of activated human PBL for 6 h. As a positive control, we detected significant induction of FASL expression. Notably, we detected an even greater increase (> 5 fold) in BIM transcription in response to OKT3 stimulation (Figure 2A). Only BCL-xL was also increased with restimulation, whereas other BCL-2 family members remained largely unchanged or slightly decreased. The expression of all three BIM protein isoforms (extra long (EL), long (L), and short (S)) also increased substantially over time with OKT3 restimulation, whether Fas blockade was applied or not (Figure 2B). Although BCL-xL protein levels also increased, the ratio of BIM:BCL-xL expression rose substantially over time, suggesting heightened Bim expression represents a \"tipping point\" for overcoming the anti-apoptotic function of BCL-xL and related proteins in driving mitochondrial depolarization. PUMAβ levels also showed a minor increase (Figure 2B). Remarkably, the quick induction of BIM upon restimulation occurred in the presence of IL-2, which is required for TCR-induced death[6]. IL-2 signaling alone can destabilize BIM mRNA or promotes BIM protein degradation via Raf/ERK or phosphoinositide kinase 3 (PI-3K) signaling pathways [25-27]. However, our results suggest the TCR restimulation overrides this signal to allow for rapid BIM upregulation. These data are consistent with previous observations indicating BIM expression can be induced upon TCR triggering in human CTL clones, depending on the agonistic peptide used [28,29]. However, these studies did not establish whether loss of BIM expression had functional consequences for TCR-induced apoptosis sensitivity, or how this related to FAS-FASL signaling.Figure 2Induction of BIM expression contributes to TCR-induced apoptosis. (A) Mircoarray analysis of designated Bcl-2 family members was performed using RNA purified from activated human PBL either untreated (0 h) or stimulated with OKT3 for 6 h. Relative expression values normalized to reference RNA from normal human PBL are shown at left, fold change following TCR restimulation is quantitated at right. (B) Activated human PBL were stimulated with OKT3 for the indicated times, and whole cell lysates were prepared and immunoblotted for the proteins indicated on the right. All three isoforms of BIM (extra-long (EL), long (L), short (S)) were detected. Spot densitometry analysis of the ratio of BIM-EL to BCL-xL (normalized to β-actin loading control) is plotted below. (C) Activated human PBL were transfected with nonspecific (NS) or Bim-specific siRNA, rested 4 days, and then restimulated for 24 h with increasing doses of OKT3 in the presence or absence of SM1/23. Percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity (relative to NS alone) were statistically significant for each dose of OKT3 (p < 0.04), except for SM1/23 treated NS cells at 1 μg/ml. (D) Lysates from cells transfected in (C) were immunoblotted for BIM as in (B). β-actin serves as a loading control. (E) Average extent of TCR-induced apoptosis inhibition (relative to NS siRNA alone) is shown for each condition described in (D) for 6 different normal donor PBL tested.To definitively test whether BIM contributes to the TCR-induced apoptosis signal, we silenced BIM expression by RNA interference (RNAi) in activated PBL and restimulated them with OKT3 with or without FAS blockade. Knockdown of BIM expression significantly reduced the sensitivity of activated PBL to TCR-induced death (Figure 2C). Control immunoblots showed that BIM expression was silenced effectively in cells that received BIM-specific siRNA both before and after restimulation (Figure 2D, Additional File 2). As noted above, FAS blockade also partially rescued cells from death in these experiments, and had an additive protective effect when BIM expression was reduced (Figure 2C). The protective effects of BIM suppression and Fas blockade were noted in multiple human donors (Figure 2E). Knockdown of FAS associated death domain (FADD) rescued cells from TCR-induced death to a similar extent, further illustrating that death receptor signaling is only part of the apoptotic signal triggered by TCR restimulation (Additional File 3). In addition, knockdown of PUMA also provided some protection from TCR-induced death (Additional File 4), although this effect was variable in different donors tested. Collectively, our data definitively shows that intrinsic apoptosis mediators, particularly BIM, are required for optimal apoptosis triggered by TCR re-engagement separate from extrinsic FAS-induced apoptotic signals.Relative contribution of BIM in CD4+ versus CD8+ TCR-induced deathWe next tested whether BIM induction played a role in TCR-induced death of both CD4+ and CD8+ T cells. Purified CD4+ and CD8+ T cells sorted from activated PBL were transfected with NS or BIM-specific siRNA and tested for sensitivity to OKT3-induced death. Whereas Fas blockade alone substantially rescued the apoptosis of purified CD4+ T cells, knockdown of Bim expression had little effect (Figure 3A). Conversely, CD8+ T cells relied on both FAS and BIM for TCR-induced apoptosis signaling. Although BIM expression was consistently higher in CD4+ T cells compared to CD8+ T cells from multiple donors (Figure 3B), BIM induction from steady state levels was as good or better in CD8+ T cells upon restimulation (Additional Files 2 &5). We cannot rule out that residual BIM expression in CD4+ T cells following BIM siRNA transfection contributed to the Fas-independent of apoptosis observed. However, other experiments revealed that BIM knockdown using the same siRNA provided greater protection from IL-2 withdrawal apoptosis in CD4+ T cells (Additional File 6), suggesting BIM levels could be sufficiently depleted to hinder BIM-dependent death. Collectively, the data suggests that human CD8+ T cells rely on BIM more extensively for TCR-induced deletion than CD4+ T cells, which are largely dependent on FAS signaling. This idea agrees with landmark studies that implicated FAS in TCR-induced apoptosis, which focused primarily on CD4+ T cell lines or clones from humans or mice [15-18]. Moreover, our data potentially explain new studies suggesting BIM drives Ag-specific CD8+ T cell deletion in establishing peripheral tolerance in both mice and humans [30,31].Figure 3Bim is important for TCR-induced apoptosis of CD8+ T cells. (A) CD4+ or CD8+ T cells purified from activated human PBL were transfected with NS or Bim-specific siRNA, rested 4 days, then restimulated with increasing doses of OKT3 in the presence or absence of SM1/23. Percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity were statistically significant for SM1/23 treated CD4+ cells (NS and Bim) compared to NS cells alone (p < 0.007), except for SM1/23 treated NS cells at 1 μg/ml OKT3 (p < 0.07). Differences in apoptosis sensitivity for CD8+ T cells (relative to NS alone) were all statistically significant (p < 0.05). (B) Lysates from cells transfected in (A) were immunoblotted for BIM. β-actin serves as a loading control.Bim and Fas cooperate in TCR-induced apoptosis of murine T cellsIn light of our findings in human T cells, we re-examined TCR-induced death in murine T cells. Surprisingly, we observed that activated splenic T cells from Fas-deficient lpr mice showed only minor resistance to anti-CD3-induced death induced by restimulation, whereas bim knockout mice showed no difference in sensitivity compared to WT cells (Figure 4A). We also tested for Bim induction in restimulated WT and lpr T cells in the presence of IL-2. Consistent with data in human T cells, activated mouse T cells (WT or lpr) showed a clear increase in BimEL expression after 6 hours of restimulation (Figure 4B). We also detected a change in the migration of BimEL and BimL isoforms, suggesting post-translational modifications may affect of bim function in mice, perhaps via phosphorylation.Figure 4Fas and Bim cooperate in driving TCR-induced apoptosis of murine T cells. (A) Activated splenic T cells from wild-type (WT), lpr, or bim-/- mice were restimulated with platebound anti-CD3 for 24 h. Percent cell loss was calculated in triplicate by PI exclusion. (B) Lysates from splenic T cells from the indicated genetic backgrounds left untreated or restimulated with platebound anti-CD3 for 6 h were immunoblotted for Bim isoform expression. β-actin serves as a loading control; asterisk indicates non-specific band. (C, D) CD4+ and CD8+ T cells were purified from activated WT, lpr, or bim-/- splenocytes and restimulated with platebound anti-CD3 for 24 h. Percent cell loss was calculated in triplicate by PI exclusion. (E) Splenic T cells from WT or lpr mice were stimulated for 48 h with platebound anti-CD3/anti-CD28, washed, and transfected with NS or Bim-specific siRNA. Three days post-transfection, cells were restimulated with 100 ng/ml platebound anti-CD3; percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity (relative to NS-treated WT cells) were statistically significant (p < 0.04). Lysates made from cells three days post-transfection were assessed for Bim knockdown by immunoblotting, right.Next, we reasoned that differences in apoptosis sensitivity caused by loss of Fas or Bim may differ in CD4+ and CD8+ T cell cultures, as noted in for human T cells. Therefore, we assayed TCR-induced apoptosis sensitivity in purified CD4+ and CD8+ T cells from WT, lpr, and bim-/- mice. As expected from previous reports, CD4+ lpr cells showed a profound defect in restimulation-induced death (Figure 4C). This concurred with our results in human CD4+ T cells using Fas blocking Ab (Figure 3A), indicating Fas is necessary for CD4+ T cell restimulation apoptosis. In contrast, there were no differences in CD8+ T cell death between restimulated WT and lpr cells, explaining the cumulatively minor rescue of TCR-induced death in total splenic T cells when Fas is absent. Furthermore, genetic ablation of bim had little protective effect for activated CD4+ T cells upon TCR restimulation, and no discernible effect on apoptosis in CD8+ T cells (Figure 4D).We hypothesized that loss of Bim from development, through germline gene ablation, may permit T cells to \"compensate\" accordingly via enhanced expression or function of pro-apoptotic molecules. Therefore, we acutely silenced Bim using RNAi in activated WT and lpr T cells. Knockdown of Bim significantly protected activated WT and lpr T cells from apoptosis induced by 100 ng/ml anti-CD3 stimulation (Figure 4E, left panel), demonstrating that Bim can play a prominent role in this apoptosis pathway. This effect was also noted in purified CD4+ and CD8+ T cell populations (data not shown), even though loss of Fas alone reduced sensitivity only in CD4+ T cells again as expected. Control blots showed that Bim siRNA effectively suppressed Bim protein expression in both WT and lpr T cells (Figure 4E, right panel). This protective effect was less pronounced at higher doses of anti-CD3 stimulation (data not shown), suggesting stronger restimulation may override Bim siRNA effects and/or trigger alternative death effector pathways. Thus, our data suggests that Fas or Bim may partially compensate for the loss of one or the other from development in murine T cells during development.Relative BIM expression correlates with sensitivity to TCR-induced death in ALPS patientsBased on our aforementioned results, we revisited TCR restimulation-induced apoptosis in PBL derived from several ALPS patients. Similar to controls, PBL cultures from ALPS Ia patients were primarily comprised of CD8+ T cells (data not shown). Surprisingly, we found that PBL from several ALPS Ia patients displayed normal or slightly more death in response to OKT3 titration compared to normal controls, despite impaired apoptosis upon direct Fas crosslinking. (Figure 5A, Additional File 7). Similarly, T cells derived from an ALPS Ib patient harboring a dominant interfering mutation in FASL [32] were also killed effectively upon TCR restimulation (Figure 5B). Consistent with defective FASL function, TCR-induced apoptosis was unaffected by Fas blockade. These results exposed a glaring contradiction in the concept that FAS mediates most or all TCR-induced death.Figure 5Suppression of Bim expression in ALPS type IV patient causes resistance to TCR-induced death. (A) Activated human PBL from normal control donors (NC1, NC2), or 6 ALPS type Ia patients were restimulated with increasing doses of OKT3 for 24 h. Percent cell loss was calculated in triplicate by PI exclusion. (B) Activated human PBL from an ALPS type Ib patient or a normal control donor (NC) were treated as in (A). Percent cell loss was calculated in triplicate by PI exclusion. (C) Activated human PBL from normal control donors (NC1, NC2), or 6 ALPS type Ia patients restimulated with 100 ng/ml OKT3 for 0 (-) or 8 h (+), lysed and immunoblotted for BIM. β-actin serves as a loading control. Spot densitometry analysis of the ratio of BIM (EL isoform) to β-actin (normalized to NC1 untreated, dashed line) is plotted below. (D) Activated human PBL from normal control donors (NC1, NC2), an ALPS type Ia patient, and an ALPS Type IV patient (P58) were restimulated with OKT3 for 24 h. Percent cell loss was calculated in triplicate by PI exclusion. Differences in apoptosis sensitivity (relative to NC1 or NC2) were statistically significant (p < 0.01). (E) Activated human PBL as in (D) were restimulated with 100 ng/ml OKT3 for 0 (-) or 8 h (+), lysed and immunoblotted for BIM. β-actin serves as a loading control.We next assessed the relative expression of BIM before and after restimulation of PBL in ALPS Ia patients. In general, we noted higher BIM protein expression in restimulated ALPS Ia T cells relative to controls (Figure 5C). In 4/6 ALPS Ia patients, steady-state BIM expression was also elevated relative to controls. Using spot densitometry, we estimated that ALPS Ia T cells had between 30–80% more BIM protein than normal controls both before and after TCR ligation (Figure 5C, bottom panel). BIM siRNA treatment did not result in a significantly greater rescue of TCR-induced death in ALPS Ia cells compared to normal controls (data not shown), perhaps due to incomplete depletion of BIM or compensation by other mediators (e.g. PUMA). Nevertheless, elevated BIM levels in cycling T cells with defective FAS function may suggest that these T cells are \"primed\" for apoptotic deletion through a compensatory increase in BIM expression.Finally, we tested TCR-induced apoptosis in T cells derived from an ALPS Type IV patient (P58) with a gain-of-function, germline NRAS mutation that constitutively activates ERK and suppresses BIM expression. We recently demonstrated that P58 T cells are resistant to apoptosis induced by IL-2 withdrawal due to BIM suppression[12]. Remarkably, P58 T cells displayed partial resistance to TCR-induced death when compared to normal donor and ALPS Ia cells, despite comparable expression of FAS on the cell surface (Figure 5D, Additional File 8). Moreover, BIM expression was attenuated in P58 T cells and could not be rescued by TCR restimulation (Figure 5E), providing stronger evidence that BIM serves a physiologically relevant role in the restimulation apoptosis pathway, especially for CD8+ T cell homeostasis. Moreover, our data implies that relative BIM expression may represent an important determinant of TCR-induced apoptosis sensitivity, independently of FAS. However, we concede that NRAS/ERK dysregulation in P58 could alter TCR-induced death through BIM-independent mechanisms as well. Indeed, pharmacological ERK inhibitors actually provided a small but reproducible rescue of TCR-induced death in both normal and P58 T cells.The physiological function of Bim was originally revealed from characterization of Bim-deficient mice, from which T cells were profoundly resistant to lymphokine withdrawal death[8]. The pro-apoptotic function of Bim also enforces immune tolerance through thymocyte negative selection, CD8+ T cell cross tolerance, and the regulation of antigen presenting cells including B cells and dendritic cells[23,33-35]. Here we demonstrate that BIM also plays a significant role in TCR-induced death of activated human T cells, working in tandem with FAS signaling as a separate signal to kill T cells. This provides a new mechanism besides the cleavage of BID for an extrinsic signal to activate the intrinsic mitochondrial death program. This paradigm may be distinct from Bim-dependent \"activated T cell death\" described by Hildeman et al. in mice challenged with a single dose of superantigen [36], which may be interpreted as predominantly cytokine withdrawal apoptosis, not restimulation-induced death with repeated Ag dosing. On the other hand, the marked accumulation and persistence of Bim-deficient murine CD8+ T cells in chronic viral infection models could be connected to failed deletion in response to repeated TCR stimulation [37,38].Our results show that direct signals from the TCR program T cells to die through Bim, which is fundamentally different from the secretion of death cytokines such as FasL that engage external death receptors. This has some interesting implications. First, it may be advantageous in conditions where Fas may not be effective. For example, Bim has a greater influence in CD8+ T cells that can utilize FasL:Fas as a calcium-independent cytolytic mechanism against infected target cells and therefore may be inured to its lethal effects. Second, the direct molecular connection inside the cell may make the Bim pathway more efficient. Careful investigation of the temporal effects of killing after TCR engagement may reveal differences between Fas and Bim effectiveness. Third, as Bim expression is extensively regulated post-translationally, the fact that translation inhibitors only partially block TCR-induced death could indicate there is a direct death pathway entrained to TCR restimulation that does not require new protein synthesis [17]. Finally, pro-apoptotic mediators like Bim or Puma acting at the convergence of TCR and CWA may help to restrain these pathways at a focal point for tight control of those T cells escape death and emerge as memory T cells.Recently, three groups reported that loss of both Bim and Fas in mice results in massive lymphadenopathy/splenomegaly, early onset of SLE-like autoimmune manifestations, and even greater accumulation of antigen-specific CD8+ T cells upon chronic viral infection [39-41]. These experiments reprised earlier work that obtained very similar results when transgenic Bcl-2 overexpressing mice were crossed onto an lpr background [42,43]. However, their general conclusions still emphasized the traditional model, reiterated in an accompanying review, that Fas and Bim control T cell homeostasis through two distinct pathways: restimulation-driven versus IL-2 withdrawal-induced apoptosis, respectively[5,7,19]. Our study illustrates that death of activated T cells via Fas or Bim are not mutually exclusive pathways, as both can operate in IL-2 dependent TCR-induced apoptosis. During infections this combination of potent extrinsic and intrinsic signals may act to ensure rapid and efficient killing of hyper-responsive or cross-reactive autoimmune T cells upon repeated antigen encounter, thus preventing immunotoxicity and maintaining peripheral tolerance. Another intriguing possibility relates to the potential of Bim and Fas to partially compensate for one another in driving TCR-induced apoptosis. This applies to situations where either gene function is lost from development, such as in lpr or bim-/- mice, and may explain why only acute knockdown of Bim resulted in significant reduction of TCR-induced apoptosis in murine T cells in vitro. The idea that Bim participates in ensuring T cell homeostasis both during and after effector T cell responses may also explain why Bcl-2 Tg lpr mice described years ago have strikingly worse lymphocyte accumulation compared to either Bcl-2 Tg or lpr mice alone[43]. Our results provide a new interpretation of the mouse studies by revealing that the infection-induced derangement of T cell homeostasis caused by Bim-deficiency could be accounted for by an impairment of both intrinsic and extrinsic apoptosis. It is also notable that ALPS patients show wide variability in conventional CD3+ T cell numbers, with a substantial fraction showing no increases. By contrast, the fraction and absolute number of \"double negative\" (CD4-CD8-) α/β T cells are invariably elevated [44]. This may reflect that alternative effectors such as BIM could preserve equipoise in the conventional T cell compartment.In humans, our data are consistent with previous studies suggesting that TCR-induced death involves multiple effector molecules, and clearly includes components other than FAS or BIM that remain to be elucidated[22]. We have previously noted a role for tumor necrosis factor-alpha (TNF-α) in this process for murine CD8+ T cells[21]; however, blockade of this pathway in human T cells had little demonstrable effect (data not shown), which requires further exploration. A recent paper from Mateo et al. implicated perforin and cytotoxic granules in the execution of TCR-induced death, particularly for ALPS Ia patient cells[45]. Other inputs implicated in control of AICD sensitivity, including NF-κB regulation through HPK-1 or generation of reactive oxygen species (ROS), likely relate more to the regulation of FasL or Bim expression[46,47]. However, we are studying patients with impaired TCR-induced apoptosis despite normal induction of FASL and BIM, and normal apoptosis triggered by FAS ligation or IL-2 deprivation (A.L. Snow, unpublished data). Insights gleaned from such patients may further advance our understanding of the biochemical complexity and physiological relevance of apoptosis in different immune cell compartments. Nevertheless, our current findings further elucidate FAS-independent signals for the restimulation-induced death of activated T cells via BIM induction.ConclusionAlthough Fas-FasL signaling is often considered synonymous with TCR restimulation-induced death, the data provided herein show it has a quantitatively lesser role than previously acknowledged, and support a critical role for BIM induction in the execution of antigen-driven \"extrinsic\" apoptosis. Increased BIM expression following TCR restimulation, even with a surfeit of IL-2, works in parallel to FAS signaling in driving mitochondrial depolarization, caspase 9 activation, and eventual apoptosis. Like FAS blockade, suppression of BIM induction via RNAi or increased NRAS activity in an ALPS variant patient results in partial resistance to TCR-induced death. These data build upon previous work from Sandalova and colleagues by demonstrating that BIM is indispensable for maximum sensitivity to restimulation-induced apoptosis of human T cells. More importantly, our findings revise previous models in showing that FAS and BIM both participate in eliminating activated T cells through this IL-2 dependent, propriocidal death pathway.AbbreviationsALPS: Autoimmune lymphoproliferative syndrome; FasL: Fas ligand; PBL: Peripheral blood lymphocytes; PI: Propidium iodide.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsALS and JBO designed and performed all experiments. ALS wrote the manuscript. LZ helped with activation and analysis of ALPS Type Ib cells. JKD. cared for ALPS patients and facilitated sample collection and distribution. MJL and TAF helped direct the research and contributed to final revisions.Reviewers' commentsReviewer #1, Snow et al.:Dr. Wendy Davidson, University of Maryland School of MedicineEditor: Dr. David Scott, University of Maryland School of MedicineThis manuscript examines the role of BIM in TCR restimulation-induced death in human T cells from healthy individuals and patients with ALPS. The authors show that restimulation of normal unfractionated T cell blasts results in significant upregulation of BIM expression and that reactivation-induced cell death (AICD) is reduced when FAS/FASL interactions are blocked with antagonist Ab or BIM expression is decreased by siRNA. Blockade of both pathways appeared to have additive effects suggesting that both BIM- and FAS-mediated death pathways contribute to AICD. Further analysis of T cell subsets showed that although CD4+ T cell blasts express higher levels of BIM than CD8+ T cell blasts prior to and after restimulation, BIM-mediated death appears to be restricted to CD8+ T cells. Combined, the data on subsets suggest that AICD in CD4+ T cells is predominantly FAS-mediated whereas CD8+ T cells are susceptible to death induced by FAS and BIM. Additional studies with unfractionated T cell blasts from ALPS patients deficient in FAS or FASL indicated sensitivity to FAS-independent AICD, increased basal levels of BIM and increased expression of BIM post restimulation. However, attempts to block AICD using BIM siRNA were unsuccessful. As an alternative indirect approach to determine the contribution of BIM to AICD in the context of ALPS, the investigators used T cells from an ALPS type IV patient with a germline NRAS mutation resulting in constitutive activation of ERK and suppression of BIM expression. Unfractionated T cell blasts from this patient were efficiently killed by anti-FAS mAb but were relatively insensitive to TCR-induced death, suggesting that BIM might contribute to AICD.The findings with normal human CD8+ T cells are novel and consistent with recent studies in mice indicating the importance of both the BIM and FAS cell death pathways in regulating CD8+ T cell homeostasis. The data relating to the role of BIM in T cell death in ALPS patients are less compelling because of the difficulties in efficiently downregulating BIM in T cell blasts from ALPS type I patients. Further, in the ALPS type IV patient the authors assume, but do not prove, that the only way that dysregulated expression of NRAS protects T cells from restimulation-induced death is through down regulation of BIM.Critique:Major points:1. The key data shown in Figs. 2C and 3A appear to be from a single experiment utilizing cells from one individual. The investigators should indicate how reproducible the studies were and whether there was significant variation in susceptibility to BIM-mediated killing among T cells from different individuals.Author ResponseThe reviewer poses an important question. Although some variation was noted in cells from different donors, the experiments described were very reproducible in multiple replicate experiments, utilizing several human donors. In order to clarify this point, Figure 2E has been added to summarize the extent of TCR-induced apoptosis inhibition, relative to NS siRNA-transfected cells, for 6 different human donors tested.2. In Fig. 2D, the authors show that BIM expression is significantly reduced in T cell blasts after transfection with BIM siRNA. The authors do not specify whether the cell lysates came from blasts 4 days post transfection or from restimulated transfected blasts. Similar data shown in Additional File 2 indicate that western blots were performed prior to restimulation. It is imperative that the authors demonstrate that restimulation-induced upregulation of BIM is prevented in the BIM siRNA transfected blasts. Otherwise, the data cannot be interpreted.Author ResponseWe agree with the reviewer's critique, as blots shown represent cells lysed 4 days post-transfection of siRNA without restimulation. To address this point, the new Additional File 2 now demonstrates that Bim siRNA effectively suppresses BIM expression/induction in both resting and restimulated T cells.3. Data in Fig. 3A suggest that BIM either does not contribute to restimulation-induced death in CD4+ T cell blasts or that the BIM death pathway is far less efficient than the FAS death pathway in inducing apoptosis in this population. Studies with Bim KO mice may help to distinguish between these two possibilities, assuming that murine CD4+ T cells reliably model human CD4+ T cells in this regard. Decreased sensitivity of BIM knockdown CD4+ blasts to IL-2 withdrawal-induced death would provide additional evidence that BIM levels are sufficiently depleted to impair BIM-dependent death.Author ResponseFollowing the reviewer's suggestion, we have added a new figure detailing several experiments performed with mouse T cells from Bim KO as well as lpr mice (new Figure 4). These data support the conclusion that CD4+ T cells are much less dependent on Bim compared to Fas for TCR-induced death, although we found that CD8+ T cells were equally sensitive to the blockade of either pathway. On the other hand, acute knockdown of Bim expression in mouse T cells rescued a substantial proportion of WT T cells, and almost all lpr T cells, from TCR-induced apoptosis. We hypothesize that Bim KO cells maintain sensitivity to TCR-induced death through a compensatory effector molecule (Fas or otherwise), such that the acute knockdown in WT cells does not allow sufficient time or the physiological context for a hypothetical compensatory mechanism to have a significant effect.We also provide new data (Additional File 6) showing that our RNAi approach provides sufficient depletion of BIM to impair BIM-dependent, IL-2 withdrawal apoptosis in CD4+ T cells. In fact, the extent of this protection was more pronounced for CD4+ T cells compared to CD8+ T cells. This contrast suggests the more prominent role of BIM in human CD8+ T cell deletion, supported by our data in Figure 3, is specific to TCR restimulation-induced death.In Fig. 2C, the data suggest that the FAS and BIM death pathways are equally effective in killing blasts enriched for CD8+ cells. However, in Fig. 3A purified CD8+ blasts appear to be more sensitive to FAS-mediated death. This difference deserves comment. The author's should also indicate whether the greater sensitivity of CD8+ blasts to FAS-mediated death is a consistent finding in healthy humans. As discussed above for the unfractionated blast populations, data showing that siRNA blocks the restimulation-induced increase in BIM expression in the purified CD4+ and CD8+ are essential.Author ResponseIn general, we cannot definitively say that purified CD8+ T cells are more sensitive to the Fas-FasL component of TCR-induced death than unfractionated PBL. The data shown in Figure 3A are representative of several experiments, although some variability in the extent of rescue by the Fas antagonist antibody SM1/23 was noted in both purified CD8+ T cells and PBL. As noted in the text, the majority of T cells found in normal donor PBL (roughly 55–80%) were CD8+ T cells following a week of cycling in IL-2. As mentioned above, Additional File 2 demonstrates that Bim-specific siRNA blocks the restimulation-induced increase in BIM expression in purified T cell subsets.4. Although T cells from ALPS patients with defects in FAS signaling are clearly susceptible to AICD and exhibit increased expression of BIM after restimulation, no conclusions can be drawn regarding the contribution of BIM in FAS-independent apoptosis in CD4+ or CD8+ T cells since the authors were unable to block BIM expression. While the ALPS type IV patient, P58, with reduced BIM levels and decreased sensitivity to AICD provides additional preliminary evidence for a role for BIM in T cell homeostasis, the authors should address the possibility that the enhanced cell survival associated with NRAS dysregulation may result from effects other than, or in addition to, BIM downregulation.In Fig. 4D, the investigators show that P58 blasts are highly sensitive to anti-FAS-induced apoptosis but do not establish whether FAS-mediated AICD is intact. Ideally, the AICD experiments should be repeated with isolated P58 CD4+ and CD8+ blasts in the presence or absence of antagonist anti-FAS Ab to determine the level of FAS-mediated apoptosis in each population. If P58 CD4+ blasts are sensitive to AICD and death is significantly inhibited by FAS blockade, the investigators will have additional evidence that BIM contributes minimally to AICD in CD4+ T cells.Author ResponseWe appreciate the authors' interpretation. Accordingly, we now provide additional commentary in the Discussion to point out that the gain-of-function NRAS mutation in P58 T cells could be altering TCR-induced death signaling through additional unidentified BIM-independent pathways. Although purified CD4+ and CD8+ T cells were not tested, TCR-induced death in P58 PBL could be partially rescued by Fas blockade to a similar extent as normal donor T cells, suggesting FasL function is intact. We also concur that enhanced BIM expression in ALPS Ia patients is suggestive, but not definitive, evidence for BIM-dependent AICD short of better knockdown experiments.Minor Points:1. In their discussion (page 14, line 10), the authors overstate the differences between their interpretation of how FAS and BIM may work to regulate T cell homeostasis and the conclusions of three other groups studying mice deficient in Bim and functional Fas.Authors ResponseWe respectfully disagree with the author's opinion in this case, as we feel it is imperative to highlight the novelty of our findings in relation to the three cited articles pertaining to Bim-deficient lpr mice. The \"preview\" article published alongside those articles in Immunity (see references in the manuscript) maintains that Fas controls antigen-stimulated or \"active\" death, and cytokine withdrawal death governed by Bim and its interactions with other Bcl-2 family members controls antigen depletion or \"passive\" death in a mutually exclusive manner. Actually our data indicate that both of these contentions are oversimplified. Ample data indicates that Fas is not the only mediator of TCR-stimulated death of activated cells illustrated in all of the experiments in this paper by the fact that substantial residual death evident even with combined Fas/Bim blockade. Moreover, our data demonstrate that Fas and Bim can both contribute to apoptosis caused by TCR restimulation of activated T cells. This provides a different molecular schema for interpreting the synergistic increase in lymphadenopathy and autoimmunity they reported resulting from combined genetic lesions of Fas and Bim. It is clear from those papers and the overview written by Green that the interpretation is confined to a model that has existed in the field for over a decade, viz Lenardo, J. Exp. Med. 183: 1071, 1996. Our new findings reveal a functional connection between the TCR-induced pathway and Bim that was previously hinted at in the literature (Sandalova et al., Hum Immunol. 2006 Dec;67(12):958–65) and not considered in the 2008 Immunity papers. This sheds new light on the described phenotype of these mouse experiments, particularly with regard to excessive CD8+ T cell lymphoproliferation, that was not considered.2. Since the difference in susceptibility of CD4+ and CD8+ T cells to BIM-mediated AICD is a novel observation, the authors should discuss this data more extensively.3. Information on the source of the SM1/23 mAb and a reference for the cell death assay need to be included in the Methods section.4. In Fig. 4B, the axis label for OKT3 concentration should read μg/ml. On page 7 there is are typos in the spelling of microarray. Data is plural.Author ResponseWe have elaborated on our discussion of differences in BIM-driven TCR-induced death of CD4+ and CD8+ T cells as recommended by the reviewer. We have also added two very recent references that demonstrate a prominent role for BIM in Ag-specific peripheral deletion of CD8+ T cells in mice and humans, in agreement with our in vitro data. The information requested is now provided in the text, and typos have been corrected.Reviewer #2, Snow et al.:Dr. Mark Williams, University of Maryland School of MedicineEditor: Dr. Neil Greenspan, Case Western Reserve UniversityRecent papers suggest that the role of Bim in classic TCR-induced AICD is not well defined. This paper addresses this question.The data in Figure 2 suggest that Bim plays a role in AICD in the T cells from normal controls, but the data from the ALPS patients in Figure 4 do not allow such an interpretation. The P58 cells could be resistant to AICD for many other reasons besides a lack of Bim. For example FasL levels are not examined or TCR signals needed to sensitize to AICD may be deficient. The Bim siRNA data in ALPS patients also should be shown (page 13).Authors ResponseWe concede that because we were unable to knockdown BIM expression adequately in ALPS Ia patient cells, data shown in Figure 5C do not conclusively show that BIM plays a more prominent role in TCR-induced death of ALPS Ia patient cells. However, elevated BIM expression noted in several ALPS Ia patients strongly suggests that other apoptosis effector molecules, including BIM, may be compensating for the loss of functional Fas in maintaining sensitivity to TCR-induced death. The striking sensitivity of ALPS cells to TCR-induced death is a surprising effect that we believe has been unequivocally demonstrated. Indeed, a similar interpretation may be derived from our new data in Bim KO and lpr T cells (see new Figure 4). Please note our response to Reviewer 1 with regard to interpreting data from P58 cells.Statistical analysis is needed for all graphs (the word \"significantly\" is used throughout the text) especially in Figure 3 to establish that Bim knockdown gives additional protection above Fas blockade.Authors ResponseWe point out that statistical analysis (paired T tests) was applied to all of the graphs shown throughout the paper, as noted in the Figure Legends (p values are included). This statistical analysis is now mentioned in the Methods section for clarification. The word \"significantly\" was applied in the manuscript with these statistical analyses in mind, referring only to differences with p < 0.05.Overall, the data support a role for Bim in AICD, albeit not as strongly as the authors suggest. More studies in which Bim expression is manipulated are necessary to support such a conclusion.Authors ResponseCurrently, direct silencing of BIM using siRNA is our most effective tool for manipulating BIM expression in primary human T cells. Unfortunately, ectopic expression of BIM in primary cells is more problematic, as cell viability is poor due to both BIM overexpression and increased cell loss during electroporation of an expression plasmid. We hope that the inclusion of new data in Figure 2E and Additional File 2 strengthens our conclusion that BIM siRNA effectively suppresses BIM expression during restimulation, translating to AICD resistance in multiple human donors tested. Furthermore, the effect of acute Bim knockdown in WT and lpr mouse T cells resembles our data in human T cells and supports our general hypothesis further.Reviewer #3, Snow et al.:Editor: Laurence C. Eisenlohr, Thomas Jefferson UniversityComments:1. In reference to the term \"propriocidal cell death\": I'm familiar with \"proprioception\" but not \"propriocidal\".Author ResponseWe employ the term \"propriocidal\", based on the Latin prefix (\"proprio\" = \"of his own accord\"), to refer to TCR-induced apoptosis as a self-regulatory form of T cell death. This has been introduced in the literature on this topic in the past: Nature 353: 858–861, 1991, Eur. J. Immunol., 23: 1552–1560, 1993. Although this term may be somewhat recondite, it is interesting that the Editor draws the intended analogy. Just as the proprioceptive system of inhibitory neurons provides negative feedback to control motor movements, we theorize that the negative feedback of propriocidal T cell death can govern the intensity of an immune response to prevailing conditions of antigen and IL-2. To avoid confusion we will clarify this term in the Introduction and Discussion.2. In reference to first sentence of \"Conclusions\" section of Abstract: This sentence is a speed bump for me. This is the first mention of IL-2 but the implication is that you see BIM upregulation under both conditions – restimulation and IL-2 withdrawal. Is the IL-2/BIM connection already published?Author ResponseAs explained in the Introduction, the connection between IL-2 withdrawal and BIM upregulation is well established in the literature. However, we emphasize our novel finding that robust BIM upregulation in the presence of IL-2 upon TCR restimulation suggests a different signal emanating from the TCR can override BIM destabilization normally conferred by IL-2R signaling.3. In reference to Introduction (pg 4, paragraph 2): This sentence seems a little off point. If I understand correctly, it is illustrating how Fas might play a role beyond T cells in controlling autoimmunity, not how TCR-induced death can be mediated via a non-Fas mechanism.Author ResponseWe agree with the reviewer's opinion, and have modified the sentence in question to emphasize that these data suggest Fas is less important for killing activated T cells in the context of repeated antigen restimulation than previously presumed. The next sentence provides further detail on this point.4. You need to confirm that the SM1/23 antibody is saturating. Have you tried a dose-response where it flattens below 100%? Does this adequately address the issue of saturating antibody, as PBL were used in both cases.Author ResponseWe have confirmed that use of SM1/23 Fas blocking Ab is saturating at the 1 μg/ml dose used throughout the study, both in primary human T cells as well as Jurkat T cell lines. Adding more than 1 μg/ml did not provide additional protection.5. In reference to sentence on pg 10 (\"IL-2 signaling normally destabilizes BIM mRNA or promotes BIM protein degradation via Raf/ERK or phosphoinositide kinase 3 (PI-3K) signaling pathways.\") Is this the right word or would \"alone\" be better?Author ResponsePer the reviewer's suggestion, we have modified the sentence to read \"IL-2 signaling alone can destabilize BIM mRNA...\"6. In reference to Bim knockdown in CD8+ T cells (Figure 3B, Additional File 3): Could you titrate your siRNA so that you get a similar level of residual Bim in CD8s and see if you get the same compromise in apoptosis rescue?Author ResponseWe appreciate the reviewer's suggestion, and will try this approach in subsequent studies. On the other hand, our experience indicates that adding more siRNA does not improve BIM knockdown and may lead to off-target effects, which is why we did not pursue this further in the context of the current study.7. Insert \"T cell\" on pg 13 to clarify the term \"cross-tolerance\".Author ResponseThe term is added as suggested, thank you.Supplementary MaterialAdditional File 1Blockade of Fas-induced apoptosis in SM1/23 treated PBL. Activated PBL were pre-treated with 1 ug/ml SM1/23 Ab prior to addition of increasing amounts of APO1.3 Ab. Percent cell loss was calculated 24 h later by PI exclusion in triplicate.Click here for fileAdditional File 2Bim siRNA effectively suppresses restimulation-induced BIM expression in activated T cells. Activated human PBL, purified CD4+ and CD8+ T cells were transfected with nonspecific (NS) or Bim-specific siRNA and rested for 4 days. Lysates were made from cells left untreated (0 h) or restimulated for 8 h with 100 ng/ml OKT3. Knockdown of protein expression was confirmed by immunoblotting.Click here for fileAdditional File 3Knockdown of FADD or Bim expression results in partial resistance to TCR-induced death. Activated human PBL were transfected with nonspecific (NS), FADD-specific, or Bim-specific siRNA, rested for 4 days, and then restimulated for 24 h with increasing doses of OKT3 in the presence or absence of Fas blocking Ab (SM1/23). Percent cell loss was calculated in triplicate by PI exclusion (left panel). Knockdown of protein expression was confirmed by immunoblotting in whole lysates 4 days post-transfection (right panel).Click here for fileAdditional File 4Knockdown of PUMA results in partial resistance to TCR-induced death. Activated human PBL were transfected with nonspecific (NS), Puma-specific, or Bim-specific siRNA, rested for 4 days, and then restimulated for 24 h with increasing doses of OKT3 in the presence or absence of Fas blocking Ab (SM1/23). Percent cell loss was calculated in triplicate by PI exclusion (left panel). Knockdown of protein expression was confirmed by immunoblotting in whole lysates 4 days post-transfection (right panel).Click here for fileAdditional File 5BIM induction in CD4+ vs CD8+ human T cells. CD4+ and CD8+ T cells were purified from activated human PBL and restimulated with OKT3 for the indicated times. Whole cell lysates were prepared, separated by SDS-PAGE, and immunoblotted for BIM isoforms or BCL-2 expression. β-actin serves as a loading control.Click here for fileAdditional File 6BIM siRNA impairs IL-2 withdrawal apoptosis in both CD4+ and CD8+ T cells. Purified CD4+ and CD8+ T cells were transfected with nonspecific (NS) or Bim-specific siRNA and rested 24 hrs in IL-2. IL-2 was removed by thorough washing, and percent cell loss was calculated 72 and 96 hrs later by PI exclusion (left panel). The percent of apoptosis inhibition afforded by BIM siRNA (relative to NS) is graphed in the right panel.Click here for fileAdditional File 7Impaired Fas-induced apoptosis in ALPS Ia patients. Activated human PBL from normal control donors (NC1, NC2), or 6 ALPS type Ia patients were treated with increasing doses of APO1.3 mAb for 24 h. Percent cell loss was calculated in triplicate by PI exclusion.Click here for fileAdditional File 8ALPS Type IV patient T cells express functional FAS. (A) Activated PBL from a normal control donor (NC, open histogram), an ALPS type Ia patient (gray), or ALPS type IV (P58, black) were stained with FITC-conjugated anti-CD95 or isotype control Ab (dashed line) and analyzed by flow cytometry. (B) Activated PBL from a normal donor (NC), an ALPS type IV patient (P58) and an ALPS type Ia patient were treated with 20 or 200 ng/ml APO1.3 mAb plus 200 ng/ml Protein A. Percent cell loss was calculated in triplicate by PI exclusion.Click here for file\n\nREFERENCES:\nNo References"
+}

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+{
+    "id": "PMC2529300",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529300\nAUTHORS: Chenchen Wang, Christopher H Schmid, Patricia L Hibberd, Robert Kalish, Ronenn Roubenoff, Ramel Rones, Aghogho Okparavero, Timothy McAlindon\n\nABSTRACT:\nBackgroundKnee Osteoarthritis (KOA) is a major cause of pain and functional impairment among elders. Currently, there are neither feasible preventive intervention strategies nor effective medical remedies for the management of KOA. Tai Chi, an ancient Chinese mind-body exercise that is reported to enhance muscle function, balance and flexibility, and to reduce pain, depression and anxiety, may safely and effectively be used to treat KOA. However, current evidence is inconclusive. Our study examines the effects of a 12-week Tai Chi program compared with an attention control (wellness education and stretching) on pain, functional capacity, psychosocial variables, joint proprioception and health status in elderly people with KOA. The study will be completed by July 2009.Methods/DesignForty eligible patients, age > 55 yr, BMI ≤ 40 kg/m2 with tibiofemoral osteoarthritis (American College of Rheumatology criteria) are identified and randomly allocated to either Tai Chi (10 modified forms from classical Yang style Tai Chi) or attention control (wellness education and stretching). The 60-minute intervention sessions take place twice weekly for 12 weeks. The study is conducted at an urban tertiary medical center in Boston, Massachusetts. The primary outcome measure is the Western Ontario and McMaster Universities (WOMAC) pain subscale at 12 weeks. Secondary outcomes include weekly WOMAC pain, function and stiffness scores, patient and physician global assessments, lower-extremity function, knee proprioception, depression, self-efficacy, social support, health-related quality of life, adherence and occurrence of adverse events after 12, 24 and 48 weeks.DiscussionIn this article, we present the challenges of designing a randomized controlled trial with long-term follow up. The challenges encountered in this design are: strategies for recruitment, avoidance of selection bias, the actual practice of Tai Chi, and the maximization of adherence/follow-up while conducting the clinical trial for the evaluation of the effectiveness of Tai Chi on KOA.Trial registrationClinicalTrials.gov identifier: NCT00362453\n\nBODY:\nBackgroundKnee osteoarthritis (KOA) is a growing problem in the elderly, resulting in pain, functional limitations, disability and decreased quality of life leading to lost productivity and increased health care costs [1,2]. The pathophysiological basis of KOA is multifaceted and includes intra-articular inflammation and collagen degradation, impaired muscle function, reduced proprioceptive acuity and the psychological traits of chronic pain [3-6]. Currently, there are neither feasible preventive intervention strategies nor effective medical remedies for the management of KOA.Over the past 2 decades, Tai Chi, a form of mind-body therapy, has spread worldwide for health and fitness [7]. Tai Chi combines deep diaphragmatic breathing and relaxation with many fundamental postures that flow imperceptibly and smoothly from one to the other through slow, gentle, graceful movements. Significant improvements have been reported in balance, strength, flexibility, cardiovascular and respiratory function, and reduction of pain, depression, anxiety and arthritic symptoms in a variety of patient populations including KOA [8].Thus, Tai Chi has the potential to become a novel, logistically feasible way of providing standardized exercises with a complementary mind-body approach to the management of KOA. The physical component provides exercise that is consistent with recommendations for OA (range of motion, flexibility, muscle conditioning, and aerobic cardiovascular exercise) [9], while the mind component has the potential to increase psychological well-being, life satisfaction, and perceptions of health [10]. These effects are especially pertinent for the treatment of older adults who have OA with knee pain and poor physical function.To date, only five randomized controlled trials (RCTs) conducted between 2000 and 2007 have compared the effect of Tai Chi with various controls in patients with OA [11-15]. The results of three RCTs suggested significant pain reduction compared to controls [11-13], but the other two found no significant changes [14,15]. Significant improvements in physical function were also reported in three RCTs compared with controls [11,13,14], but no effects were seen in the other two [12,15]. Of the two RCTs [14,15] that evaluated the effects of Tai Chi on quality of life, only one reported positive results for Tai Chi compared with controls [15]. In addition, only two RCTs reported significant differences between Tai Chi and control in improvements in flexibility or balance [11,13]. Heterogeneity of controls, different Tai Chi styles, doses and duration in addition to multiple OA sites prohibit a meaningful comparison across these trials. Furthermore, the absence of radiographic evidence of KOA as specified by the ACR criteria for OA [14], high dropout rates [13,14], small sample size [11-13,15], the lack of standardized outcome measures and short follow up [11-15] limit widespread applicability of the results from these studies.Because the overall findings from these RCTs suggest some favorable effects of Tai Chi on pain, physical function, quality of life, balance and flexibility in patients with KOA, a well designed study may be able to overcome the limitations of the previous studies and provide a more useful treatment. We hypothesize that Tai Chi may be beneficial to patients with KOA as a result of an effect on muscle strength, flexibility, pain, stress and anxiety as well as \"mind-body\" interactions. We therefore designed a 12 week trial with long term 1 year follow up to obtain data on the effects of Tai Chi on pain (as a marker of disease activity), functional independence (a marker of impairment), disability, joint proprioception and health status in elderly people with KOA.In this paper, we present the design and detailed protocol of a single-blinded, randomized controlled trial as well as a discussion of the overall challenges of conducting this trial with respect to strategies for recruitment, avoidance of selection bias, the actual practice of Tai Chi, and the maximization of adherence/follow-up. We report ways to overcome the theoretical and logistic limitations and problems of conducting such a clinical trial. The results from this trial will be reported at the completion of the study in accordance with the Consolidation of Standards for Reporting Trials guidelines [16].Methods/DesignStudy designThis study is a single-blinded, randomized, attention-controlled, clinical trial to evaluate the physical and psychological effects of Tai Chi for patients suffering from tibiofemoral KOA. Our goal is to compare the safety and effectiveness of Tai Chi with an attention control (wellness education and stretching program) in 40 patients with KOA. (The \"single-blind\" study design has been decided upon because of the inability to conceal Tai Chi assignment allocation from participants and the instructors in Tai Chi clinical trials. However, all study evaluators will be masked to treatment assignment throughout the duration of the study.)Outcome parameters compare changes in knee pain, stiffness, and physical function using the well-validated Western Ontario and McMaster University Index (WOMAC), patient and physician global assessments, lower-extremity function, knee proprioception, depression, self-efficacy, social support, health-related quality of life, outcome expectation, adherence and occurrence of adverse events. Outcome measurements are performed at baseline, every week during the intervention period, and on completion of the 12-week program, in addition to the 24 week and 48 week follow-up. Study evaluators (study rheumatologist, study coordinator, exercise physiologist and statistician) are masked to treatment assignment.The study setting is located in an urban tertiary care academic hospital (Tufts Medical Center) in Boston Massachusetts. The study has received Ethics approval from the Tufts Medical Center/Tufts University Human Investigation Review committee and will be conducted in the Clinical Research Center and Division of Rheumatology at Tufts Medical Center.Study populationThis study comprises individuals with age > 55 years, Body Mass Index (BMI) ≤ 40 kg/m2, and with knee pain on most days of the previous month during at least one of the following activities: walking, going up or down stairs, standing upright, or in bed at night [17]. Furthermore, patients must have joint crepitus, morning stiffness lasting over 30 min. Participants are also required to have positive KOA radiological signs. Radiographic entry criteria are Kellgren and Lawrence (K/L) grade ≥ 2, defined as the presence of osteophytes in the tibiofemoral compartment and/or the patellofemoral compartment, assessed on standing anterior/posterior and lateral views (American College of Rheumatology criteria) [17]. In addition, eligible participants must have a WOMAC pain subscale score (visual analog version) of > 40 (range 0 to 500). Subjects are excluded if they have: 1. prior experience with Tai Chi or other similar types of complementary and alternative medicine such as Qi Gong, yoga or acupuncture; 2. cardiovascular or other severe disease limitations precluding full participation, as determined by the patient's primary care provider; 3. any intra-articular steroid injections in the previous 3 months or reconstructive surgery on the affected knee; 4. any intra-articular Synvisc or Hyalgan injections in the previous 6 months.Patients can continue routine medications such as non-steroidal anti-inflammatory drugs (NSAIDS) and acetaminophen, and maintain their usual treatment visits with their primary care physician or rheumatologist throughout the study. The investigators record any changes made to treatment but do not change or recommend change in medical therapy.Recruitment strategiesSignificant gaps in research participation do exist among ethnic minorities and thus limit the generalizability of findings. To ensure adequate enrollment of underrepresented groups, we place advertisements in the media (radio, local television, Internet, SAMPAN – a Chinese newspaper, the Boston Metro and Boston Globe newspapers). We also use the rheumatology clinic patient database at Tufts Medical Center for identifying patients with KOA. For interested respondents, the investigators provide information about the study and administer a brief, scripted interview to determine the caller's eligibility for the trial. This screening poses questions whose predictive values for KOA are known from population-based data. The lists of applicants who screen positive on the telephone interview are provided daily to the trial staff to schedule eligibility visits.During the 5-month recruitment period, we screened 366 patients from the greater Boston area using telephone interviews from which 62 potential patients were brought into the Clinical Research Center at the Tufts Medical Center for further eligibility screening. Of these, 40 (65%) were eligible after baseline evaluation and randomized to the Tai Chi or attention control group (wellness education and stretching) (See Figure 1). The major reason for ineligibility was the absence of radiographic evidence for KOA.Figure 1Study flow chart.Strategies to maximize adherenceAdherence in clinical research is vital and can determine study quality and the validity of results. In order to maximize adherence, several procedures are performed: 1. Select a population of individuals who are both interested and reliable; 2. Screen patients using a well-designed questionnaire which in our previous studies consistently identified reliable individuals; 3. Discuss the proposed project in detail with each patient, especially the time commitment. A verbal and written commitment will be obtained from all participants, in which they state they will adhere to the program; 4. Schedule the visits at a time that is convenient for both patients and staff; 5. Recruit and assemble sufficient patients for baseline evaluation so that we have a large enough pool of patients to provide replacements if needed; 6. Perform the randomization after the baseline evaluation; 7. Provide friendly personal contact with participants under Institutional Review Board approval; 8. Organize entertaining Tai Chi and education intervention classes. Patients receive useful information that they could not reasonably expect to obtain through regular clinical care.Sample sizeOur empiric sample size is guided by numbers and outcomes of an RCT conducted at Tufts University that tested an exercise intervention among older adults with KOA [18]. That study enrolled 46 patients and randomized them to either 4-month home-based progressive strength training or an attention control group of similar duration. The results were that the strength training group experienced a 36% decrease in the WOMAC index pain subscale (the primary outcome) compared to an 11% decrease in the attention control group. Thus, the exercise group had a mean change in the WOMAC pain score of -79 (SD = 91), while the control group had a mean change of -20 (SD = 77). Based on those numbers, a sample size of 20 per group and alpha = 0.05, would have power of 60% to detect a between-group difference of -59.0. While we recognize that the study is underpowered for a definitive comparison, we are primarily concerned with gathering preliminary data in order to plan and evaluate this innovative research direction.RandomizationRandomization assignments are made using computer-generated random numbers to randomize permuted blocks of size 2 and 4 so that each block is complete. They are provided in a sealed, opaque envelope in two groups of 10 and opened upon the patient's agreement to participate. The block size is randomly assigned to minimize correct prediction of assignments while preserving approximate balance between groups. Specially designed software developed by Tufts Medical Center is used to generate the list of random numbers and treatment assignment. Randomization envelopes are not opened unless a patient meets eligibility criteria and completes the informed consent and baseline assessment. All study envelopes are saved in the individual patient's study notebook. The statistician (CS) closely supervises the preparation process with treatment assignment packets being prepared and checked on specific days. The maximum period between screening and randomization is 3 months.InterventionForty ambulatory patients with KOA are randomly assigned to receive Tai Chi (n = 20) or attention control (wellness education and stretching) (n = 20) in twice-weekly one-hour group sessions for 12 weeks (Figure 1).Tai ChiThe Tai Chi program is based on the classical Yang Style [19] with some modifications as described below. Patients participate in two 60-minute Tai Chi sessions conducted weekly for 12 weeks. Each session includes: 1. warm up and review of Tai Chi principles and techniques; 2. Tai Chi exercises; 3. breathing techniques; and 4. various relaxation methods. The teachings are carried out by a Tai Chi master (RR) who has over 20 years experience conducting Tai Chi Mind-Body exercise programs. Several modifications are developed by the Tai Chi master to achieve the physical (body) and mental (mind) goals of the study for KOA, accommodate KOA symptoms and limit dropouts. For example, we eliminate the 90 degree knee-flexor joint stance used in the traditional Tai Chi exercise that would place stress on the knees and replace it by having patients rest their backs on the wall to strengthen the quadriceps muscles and also by performing sitting and standing exercises from a chair. Patients perform 2 sets: one with the legs together and one with legs apart to further strengthen various muscles around the knee. In addition, the ability to tap into the power of the mind is developed further by having patients perform visualizations while sitting on the edge of a chair. Subjects are instructed to practice Tai Chi at least 20 minutes a day at home and encouraged to maintain their usual physical activities, but not to participate in additional new strength training other than their Tai Chi exercises.Attention control (wellness education and stretching)We use a wellness education and stretching program for the control group because this approach has been successfully used in other studies [18,20-22]. The program provides an active control for the attention being paid to the Tai Chi group. There is adequate personal contact with the subjects (for attention) with little anticipated effect on the main outcomes. The control groups also attend two 60-minute sessions per week for 12 weeks. Each session starts with 40 minutes of didactic sessions on 1) OA knowledge; 2) diet and nutrition; 3) therapies to treat OA; and 4) physical and mental health education (recognizing and dealing with stress and depression, etc). The final 20 minutes of the hourly session consists of stretching exercises involving the upper body, trunk and lower body, each stretch being held for 10 to 15 seconds. Subjects are also instructed to practice at least 20 minutes of stretching exercises per day at home. All subjects are encouraged to maintain their usual physical activities, but not to participate in additional strength training other than their class stretching exercises. Throughout the 12-week period, we track the number of and reasons for any missed sessions in both groups.MeasurementsThe Osteoarthritis Research Society currently recommends a core set of 4 domains (pain, physical function, patient's global assessment, and, for studies of at least 1 year, joint imaging) for outcome measurement for assessing KOA in clinical trials [17,23]. This core set is used in this study (except for joint imaging). Every participant is evaluated at baseline (prior to starting either intervention), after completing the intervention (12 weeks later), and at a 24 week and 48 week follow-up (see Table 1).Table 1Sequence of trial measurements for primary and secondary outcomes*VISITBaselineIntervention** (for 12 weeks)Week 12Week 24 Follow upWeek 48 Follow upTime (months)-101 – 3612Primary outcome measureWOMAC-PainXXXXXSecondary outcome measuresWOMAC-Physical functionXXXXWOMAC-StiffnessXXXXPhysicians' Global KOA SeverityXXXXPatients' Global KOA SeverityXXXXSF-36 & EQ-5D & CES-DXXXXSelf-efficacyXXXXOutcome ExpectationXXXXSocial SupportXXXXEnjoyment QuestionnaireXXXXPhysical Functional Tests***XXXXPhysical Activity QuestionnaireXXXXJoint ProprioceptionXXXXMedicationsXXXXXAdverse EventsXXXXBody Mass IndexXXXXWeekly UpdateXKnee X-RayXAdherenceXXXXFollow-up QuestionnaireXX* KOA = Knee Osteoarthritis, WOMAC = Western Ontario and McMaster Universities Osteoarthritis Index, SF-36 = Medical Outcome Survey Short-Form 36, EQ-5D = EuroQol self-repot questionnaires, CES-D = Center for Epidemiology Studies Depression index.** Randomization to Tai Chi or Attention Control intervention.*** Physical Function Tests include range of motion, standing balance, 6-minute walk, and chair standing.Global knee painThe degree of self-reported global knee pain and function is recorded using the WOMAC index [24]. The WOMAC is a self-reported instrument used to assess lower extremity pain, stiffness and physical function. The primary endpoint of the study is the change in the pain subscale of the WOMAC between baseline and the 12-week assessment.In addition, the physician provides a global score summarizing both knees using a visual analog scale version at baseline and follow up. In the baseline assessment, we use a self-reported knee-specific global pain visual analog scale (VAS) with range 0–100 mm to compare pain severity in each knee. In the event that an enrollee has identical pain scores in both knees, our statistician randomly assigns one of these to be the 'study knee' (see statistical analysis). The 'study knee' will be the knee on which primary outcome assessments are determined and used for analysis of strength outcomes.RadiographsAnterior-posterior (AP) and lateral standing knee radiographs are obtained at the initial screening examination (the baseline assessment) using the Framingham study protocol [25] and are screened by the study rheumatologist (RK) for acceptance criteria. AP knee radiographs are also obtained with the patient in the fully extended standing position and lateral images with the patients in the supine position with the knee in 30 degrees of flexion, as previously suggested [26,27]. The study rheumatologist (RK) and a musculoskeletal radiologist independently screen the radiographs using the Kellgren and Lawrence (K/L) grading system for global tibiofemoral radiographic severity, and discrepancies are resolved with a third reviewer (TM). The K/L score is determined for each knee compartment based on osteophyte formation, joint space width, and subchondral bone scleroses [28]. All scores reported are for the most severely affected knee (study knee).Knee examinationKnee examination is performed at baseline and during each follow up visit. The study rheumatologist (RK), who is blinded with respect to the patient's treatment assignments, assesses the presence and severity of the knee joint abnormalities relevant to KOA. The knee examination components were selected based on recent data demonstrating the potential for reproducibility across examiners [27]. The study rheumatologist examines for swelling and tenderness of the knee joint and asks each subject to rate pain on a scale of none, mild, moderate or severe. The examination sequence is as follows: 1. Palpate study knee joint swelling (bulge sign, balloon test and patellar tap); 2. Palpate for patella tendon tenderness; 3. Palpate for patella tenderness; 4. Palpate for anserine bursa tenderness; 5. Palpate for popliteal space tenderness; 6. Rate pain in the knee/knee joint when limb is in motion; 7. Scale the general crepitus sign as none, fine or coarse; 8. Palpate for tibiofemoral tenderness laterally; 9. Palpate for tibiofemoral tenderness medially. Evidence of inflammation (e.g. joint effusion), joint deformity, and joint contractures will also be noted. Assessment of the patient's global status is also performed.Range of motion (flexibility)The passive range of motion for both knees (at full extension and flexion) is measured with a plastic goniometer (Whitehall Manufacturing, model G300,) marked in 1 degree increments. Patients are positioned on their sides with the lower extremity to be examined resting on a stabilizing board. The fixed arm of the goniometer is placed to align with the femur, pointing towards hip joint and the other arm in alignment with the fibula. With one hand holding the goniometer and supporting the leg, taking care to maintain axis of goniometer with knee axis, patients are asked to flex and extend their leg as far as they can. The maximal degrees of full flexion and extension are then read and recorded.Knee joint proprioceptionKnee proprioception, which is reduced in KOA [29], is measured using a Biometrics™ electrogoniometer with an ADU301 angle display unit during each assessment visit. Three test angles (30, 45 and 60 degrees) are evaluated with each subject in a sitting position taken as neutral (0 degree). The electrogoniometer is placed longitudinally in alignment with the femur and tibia with a double-sided medical tape and used to determine each of the three test angles. Patients are first shown one of the angles, which is held for a few seconds, then they are asked to close their eyes and attempt to reproduce the angle; this is repeated for all three test angles.Physical performancePhysical performance assessments include the timed stand, standing balance and 6 minute walk tests. Timed stand tests measure time taken to complete ten full stands from a sitting position [30]. This is a measure of lower extremity muscle power. Patients are instructed to complete chair stand time as quickly as possible and are timed to the nearest 0.001 seconds. The same chair is utilized for testing before and after the interventions. Patients begin the chair stand seated with their arms folded across their chests, then rise to a standing position and sit back down with their back against the back rest of the chair. The test is completed when the patient stands for the 10th repetition. Chair stand time is recorded using the best (lowest) score of 2 trials.The standing balance tests include tandem, semi-tandem, side-by side, and one-legged stands. Patients are asked to maintain each position for 30 seconds. For each task, the research staff first demonstrates the task, asks the patient if they feel comfortable and ready and then supports the patient while positioning themselves [31-33]. One point is given if they exceed 30 seconds and none if they can not do or do not attempt the test.The six minute walking test is a reliable measure of functional exercise capacity [34]. Patients are asked to walk as fast and as far as possible within the 6-minute period and are accompanied by the research staff using a wheel measure (Redi measure, Redington, Windsor, CT) that measures distance covered in inches. Patients are given verbal encouragement throughout the 6 minutes and are informed of the remaining time throughout the 6 minutes. The distance covered at the end is noted and recorded.Health Related Quality of Life (HRQL)HRQL assessments are made using the Medical Outcome Study Short Form 36 (SF-36) [35], and the EuroQol (EQ-5D) [36] instruments. The SF-36 measures 8 domains: physical functioning, role-physical, bodily pain, general health, vitality, social function, emotional health and mental health. The EQ-5D measures 5 dimensions: mobility (disability), self-care (disability), usual activities (handicap), pain/discomfort (impairment), and anxiety/depression (impairment). There is also a visual analogue thermometer rating scale to evaluate the overall patient perception of health on a 0 to 100 scale. Higher scores indicate a better health state.DepressionThe Center for Epidemiology Studies Depression (CES-D) index is used to assess depressive symptoms [37]. It includes a 20-item Likert-type scale with scores ranging from 0 to 60. Higher scores indicate greater dysphoria.Outcome expectationOutcome expectations are beliefs that carrying out a specific behavior such as physical activity will lead to a desired outcome. The brief outcome expectations scale (OES) [38] contains questions that ask about physical and mental benefits and will be used to assess outcome expectations. The measure is scored by summing the ratings for all the items and dividing by 9 to get the average of all 9 items. Scores can range from 1 to 5, with 1 indicating low outcome expectations for the exercise and 5 suggesting high outcome expectations.Self-efficacySelf-efficacy is important for individuals to adopt and maintain a program of regular physical activity. It is assessed using the self-efficacy instrument developed by Marcus et al [39]. The patient rates his/her confidence of being physically active in different types of situations on a 5-item scale with responses ranging from \"not at all confident\" to \"extremely confident\". The total score is computed by calculating the average of all 5 questions. A higher score indicates greater self-efficacy.Social supportThis is assessed using the social support for physical activity scale adapted from Cohen and colleagues [40]. Thirteen questions rated from 0 to 5 were used to assess the influences of family and friends on patients in the last 3 months as they performed regular physical activity. Higher scores reflect more perceived social support from these individuals.Physical Activity EnjoymentThe Physical Activity Enjoyment Scale adapted from Kendzierski and DeCarlo [41] is used to evaluate enjoyment of physical activity. It is a self-administered 18-item, Likert scale used by patients to rate their current feelings about physical activity. The total scale score, computed after recoding, is obtained by summing the scores of all the items. High scores correspond to increased enjoyment, while low scores correspond to little enjoyment.AdherenceParticipants' attendance is monitored during each session for 12 weeks for both interventions via signing of attendance sheets. Patients are also asked to maintain daily Tai Chi or stretching exercise activity logs during the 12 week intervention and are encouraged with phone calls from the research staff to continue throughout the follow up period. At the 24 and 48 week visit, the research staff will ask about the average number of times per week and the number of minutes per session the subject practices at home.SafetyStudy patients are monitored weekly for the occurrence of adverse events defined as any undesirable experience during the duration of the study. Lack of effect of Tai Chi or stretching/education is not considered an adverse event. Patients are monitored weekly to determine whether an adverse event has occurred. All adverse events are recorded on an adverse event case report form.Statistical AnalysisThe primary outcome is the measurement of change in knee pain between baseline and 12 weeks. Secondary outcomes are measurements of change at 24 weeks and 48 weeks. These will be analyzed both as individual time points and in longitudinal analyses. Analyses will be intention-to-treat with secondary analyses based on completers.We will also explore a knee-based approach to the study that will use linear mixed models to adjust for correlations in outcome between knees. The most disabled knee will be used as the unit of analysis. One knee will be chosen at random if two knees are equally affected. Considering both knees would require accounting for the correlation between knees and for the presence of disability in only one knee for many participants. However, we will characterize individuals on the basis of unilateral vs. bilateral KOA and explore any possible influences of this characterization in secondary analyses.For the patients who withdraw during the study, every effort will be made to complete a follow-up WOMAC and clinical knee examination at the time of withdrawal. If a withdrawal assessment cannot be made, we will carry forward the last assessment of knee pain, physical function or as a conservative assessment to bias towards the null hypothesis.We will also perform a cost-effectiveness analysis to evaluate whether Tai Chi is a cost-effective therapy for patients with KOA. The costs will be estimated from health care utilization reported by subjects at the end of the trial using the Health Assessment Questionnaire that includes medication usage, hospitalization and health care professionals' visits. The differences in direct and indirect health care cost between the Tai Chi and attention control (wellness education and stretching) groups will also be compared.DiscussionIn this article, we present the challenges of designing a randomized controlled trial with long-term follow up. The challenges encountered in this design are: strategies for recruitment, avoidance of selection bias, the actual practice of Tai Chi, and the maximization of adherence/follow-up while conducting the clinical trial for the evaluation of the effectiveness of Tai Chi on KOA.A total of 40 eligible patients, 20 with Tai Chi and 20 with an attention control (wellness education and stretching) have already completed the study including 3 month recruitment, 12 week intervention, as well as 24 and 48 week follow-up at the Tufts Medical Center. We will perform analysis of the data and report the findings. Therefore, this project will provide important preliminary data on physical and psychological effects of Tai Chi for KOA. It will establish rigorous methods for future research for testing the mechanisms by which Tai Chi may influence pain, disability, and health related quality of life in people with KOA.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsCW obtained funding for the study. CW, TM, PH, RoR, CS, RK, and RaR designed the randomized controlled trial. CW, TM, PH, CS, RK and RaR conducted the research. CW and AO wrote the first draft of the manuscript. CW, TM, CS, PH, RoR, AO, RK and RaR participated in the revision of subsequent draft. All authors approved the final version of the manuscript. None of the authors declared any conflicts of financial interest.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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batch_10/PMC2529304.json

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+{
+    "id": "PMC2529304",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529304\nAUTHORS: Mauro Prato, Valentina Gallo, Giuliana Giribaldi, Paolo Arese\n\nABSTRACT:\nBackgroundIt has been shown previously that human monocytes fed with haemozoin (HZ) or trophozoite-parasitized RBCs displayed increased matrix metalloproteinase-9 (MMP-9) enzyme activity and protein/mRNA expression and increased TNF production, and showed higher matrix invasion ability. The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ) and trophozoites on production of IL-1beta and MMP-9 activity and expression. The second aim was to find out which component of HZ was responsible for the effects.MethodsNative HZ freshly isolated from Plasmodium falciparum (Palo Alto strain, Mycoplasma-free), delipidized HZ, beta-haematin (lipid-free synthetic HZ), trophozoites and control meals such as opsonized non-parasitized RBCs and inert latex particles, were fed to human monocytes. The production of IL-1beta by differently fed monocytes, in presence or absence of specific MMP-9 inhibitor or anti-hIL-1beta antibodies, was quantified in supernatants by ELISA. Expression of IL-1beta was analysed by quantitative real-time RT-PCR. MMP-9 activity and protein expression were quantified by gelatin zymography and Western blotting.ResultsMonocytes fed with HZ or trophozoite-parasitized RBCs generated increased amounts of IL-1beta and enhanced enzyme activity (in cell supernatants) and protein/mRNA expression (in cell lysates) of monocyte MMP-9. The latter appears to be causally related to enhanced IL-1beta production, as enhancement of both expression and enzyme activity were abrogated by anti-hIL-1beta Abs. Upregulation of IL-1beta and MMP-9 were absent in monocytes fed with beta-haematin or delipidized HZ, indicating a role for HZ-attached or HZ-generated lipid components. 15-HETE (15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid) a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis was identified as one mediator possibly responsible for increase of both IL-1beta production and MMP-9 activity.ConclusionResults indicate that specific lipoperoxide derivatives generated by HZ may play a role in modulating production of IL-1beta and MMP-9 expression and activity in HZ/trophozoite-fed human monocytes. Results may clarify aspects of cerebral malaria pathogenesis, since MMP-9, a metalloproteinase able to disrupt the basal lamina is possibly involved in generation of hallmarks of cerebral malaria, such as blood-brain barrier endothelium dysfunction, localized haemorrhages and extravasation of phagocytic cells and parasitized RBCs into brain tissues.\n\nBODY:\nBackgroundPhagocytosis of haemozoin (HZ, malarial pigment) or HZ-containing trophozoites alters functionality of human monocytes and macrophages. Monocyte ability to perform oxidative burst is compromised [1], bacterial killing abolished [2], antigen presentation altered [3], and ability to differentiate to functional dendritic cells disturbed [4]. Moreover, HZ-laden monocytes produce increased amounts of peroxidation products of polyunsaturated fatty acids (PUFAs) [5] and stimulate generation of several cytokines, such as TNF, IL-1beta, MIP-1alpha and MIP-1beta [6,7].It has been shown [8] that HZ/trophozoite-fed human monocytes produced increased amounts of TNF and upregulated mRNA/protein expression and activity of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme which degrades matrix proteins [9,10] and sheds TNF and IL-1beta from cell-bound precursors [11,12]. As TNF induces the synthesis of MMP-9 [13], ingested HZ was found to generate a TNF-driven positive feedback loop enhancing production of TNF and activity of MMP-9, both blocked by a specific inhibitor of MMP-9.Here it is shown that HZ/trophozoite-fed human monocytes generated increased amounts of IL-1beta and enhanced expression and activity of MMP-9. The latter appears to be causally related to enhanced IL-1beta production, as both expression and activation were abrogated by anti-hIL-1beta Abs. It is also shown that upregulation of IL-1beta and MMP-9 was absent in monocytes fed with beta-haematin (lipid-free synthetic HZ) or delipidized HZ, indicating a role for HZ-generated lipid components. 15-HETE (15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid), a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis [5] was identified as one mediator possibly responsible for increased IL-1beta production and MMP-9 activity.MethodsMaterialsAll materials were from Sigma-Aldrich, St Louis, MO, unless otherwise stated. Cell culture media RPMI 1640, Macrophage-SFM medium, TRIzol, M-MLV, oligo-dT, sense and anti-sense primers, Platinum Taq DNA Polymerase were from Invitrogen, Carlsbad, CA; Panserin 601 monocyte medium was from PAN Biotech, Aidenbach, Germany; recombinant human (rh)IL-1beta, blocking anti-human (h)IL-1beta antibodies and Merck's inhibitor I, (N-hydroxy-1-(4-methoxyphenyl)sulfonyl-4-(4-biphenylcarbonyl)piperazine-2-carboxamide), a specific inhibitor of MMP-9/MMP-13 activity, were from Merck, Darmstadt, Germany; ELISA kit for IL-1beta assay and 15-HETE were from Cayman, Ann Arbor, MI; anti-D IgG were from Immuno AG, Vienna, Austria; Percoll was from Pharmacia, Uppsala, Sweden; Dynabeads M-450 CD2 Pan T and M-450 CD19 Pan B were from Dynal, Oslo, Norway; Diff-Quik parasite stain was from Baxter Dade AG, Dudingen, Switzerland; sterile plastics were from Costar, Cambridge, UK; bicinchoninic acid protein assay was from Pierce, Rockford, IL; anti-MMP-9 monoclonal antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA; DNA-free kit was from Ambion, Austin, TX; Beacon Designer 2.1 software was from Premier Biosoft International, Palo Alto, CA; dNTPs were from Applied Biosystem, Foster City, CA. 4-hydroxynonenal (HNE) was from Biomol, Plymouth Meeting, PA. Beta-haematin (synthetic HZ) was prepared according to the Slater et al [14] procedure, modified as indicated [15].Cultivation of Plasmodium falciparum and isolation of trophozoite-parasitized RBCs and native or delipidized HZPlasmodium falciparum parasites (Palo Alto strain, Mycoplasma-free) were kept in culture as described [4]. HZ and trophozoite-parasitized RBCs (trophozoites) isolated from cultures during the first two days after infection of RBCs were added to schizonts (multinucleated parasite form). After centrifugation at 5,000 g on a discontinuous Percoll-mannitol density gradient, native HZ was collected from the 0–40% interphase and trophozoites/schizonts from the 40–80% interphase [4]. Native HZ was washed five times with 10 mM HEPES (pH 8.0) containing 10 mM mannitol at 4°C and once with PBS, and stored at 20% (vol/vol) in PBS at -20°C. For delipidized haemozoin, lipid extraction was performed as previously reported [5]. After isolation, HZ and trophozoites enriched to 95–97% parasitaemia were washed twice and reincubated in RPMI 1640 for 1 h at 37°C before opsonization and phagocytosis.Preparation and handling of monocytesHuman monocytes were separated by Ficoll centrifugation from freshly collected buffy coats discarded from blood donations by healthy adult donors of both sexes provided by the local blood bank (AVIS, Associazione Volontari Italiani Sangue, Torino, Italy) [1]. Separated lymphomonocytes were resuspended in RPMI 1640 medium and plated on wells of six-well plates. Each well received 2 ml of cell suspension containing 8 × 106 cells/ml in RPMI 1640. The plates were incubated in a humidified CO2/air-incubator at 37°C for 60 min. Thereafter, non-adherent cells were removed by three washes with RPMI 1640 and adherent cells reincubated at 37°C overnight in RPMI 1640. Shortly before starting phagocytosis, wells were washed with RPMI 1640 and Macrophage-SFM medium added (2 ml/well). Adherent cells prepared by this method were detached from the plates by scraping, stained with specific antibodies and analysed on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). As an average, monocytes (CD14+ cells) were 63.8 ± 5.7%, lymphocytes 36.2 ± 5.7% (mean values ± SD, n = 6) of all mononuclear cells. For selected experiments, lymphomonocytes were separated by Ficoll centrifugation from fresh buffy coats (see above) and monocytes immunopurified by depletion of non-monocytic cells from lymphomonocytes. Dynabeads M-450 CD2 Pan T and M-450 CD19 Pan B (Dynal) were added to the lymphomonocytes in a 2:1 ratio for 20 min at 4°C. B and T lymphocytes were removed by biomagnetic separation as specified by the manufacturer. The remaining monocytes were washed twice and resuspended in Macrophage-SFM medium. By this method monocytes (CD14+ cells) were 73.6 ± 9.5% pure, (mean values ± SD, n = 6, range 62–89.2%).Phagocytosis by adherent monocytes of opsonized trophozoites, native or delipidized HZ, beta-haematin, nonparasitized opsonized RBCs and latex particlesTo each well of a six-well plate with approx. 1 × 106 adherent monocytes, 50 μl trophozoites (10% haematocrit), native or delipidized HZ (120 nmoles HZ haem, an amount comparable to 50 μl trophozoites on haem content basis), 50 μl beta-haematin (120 nmoles haem), 50 μl anti-D IgG-opsonized RBCs (10% haematocrit) and 50 μl amine-modified, red-fluorescent latex particles (2.5% solids, diameter 0.105 μm) were added. Trophozoites, native and delipidized HZ, beta-haematin and latex particles were opsonized with fresh autologous serum, and nonparasitized RBCs were opsonized with anti-D IgG as indicated [1,5]. After opsonization, all phagocytic meals were suspended in Macrophage-SFM medium. The plates were centrifuged at low speed for 5 seconds to start phagocytosis and incubated in a humidified CO2/air-incubator at 37°C for 3 hours. This time period maximized phagocytosis and was not sufficient to induce haem-oxygenase-mediated degradation of ingested haem [16]. Thereafter, non-ingested cells, HZ, latex and beta-haematin particles were removed by four washes with RPMI 1640. The plates were then incubated in a humidified CO2/air-incubator at 37°C for the indicated times. In selected experiments, cells were incubated with rhIL-1beta (20 ng/ml), blocking anti-hIL-1beta antibodies (30 ng/ml) or Merck's inhibitor I, a specific inhibitor of MMP-9/MMP-13 activity (4 ng/ml) for 48 h.Assay of IL-1beta productionAfter termination of phagocytosis, monocytes were further incubated with Panserin 601 monocyte medium in a humidified CO2/air-incubator at 37°C for 48 h in presence (4 ng/ml) or absence of Merck's inhibitor I, a specific inhibitor of MMP-9/MMP-13 activity. The level of active soluble IL-1beta was assayed in monocyte supernatants by ELISA. A standard calibration curve was generated with rhIL-1beta, according to the manufacturer's instructions.Assay of MMP-9 activity by gelatin zymographyAfter termination of phagocytosis, monocytes were further incubated with Panserin 601 monocyte medium in a humidified CO2/air-incubator at 37°C for 48 h. Thereafter, the activity of MMP-9 was evaluated by gelatin zymography in the cell supernatants as indicated [8,17,18]. Supernatants were loaded on 8% polyacrylamide gels containing 0.1% gelatin under non-denaturing and non-reducing conditions. Following electrophoresis, gels were washed and incubated for 18 h at 37°C in a collagenase buffer. Densitometric analysis of the bands considered to reflect total enzymatic activity of MMP-9, was performed using a computerized densitometer (Chemidoc, Biorad, Hercules, CA) with activity presented in relative units compared to background.Assay of MMP-9 protein expression by western blottingAfter termination of phagocytosis, monocytes were further incubated with Panserin 601 monocyte medium in a humidified CO2/air-incubator at 37°C for 48 h. Thereafter, cells were washed and lysed at 4°C in lysis buffer containing (mM): NaCl, 300; Tris, 50; 1% (vol/vol) Triton-X100; protease and phosphatase inhibitors: pepstatin, 50 ng/ml; leupeptin, 50 ng/ml; aprotinin, 10 μg/ml. The protein content in the lysate was measured by the bicinchoninic acid assay and 12 μg protein/lane were added to the loading buffer. The lysates samples were loaded on 8% polyacrylamide gels under denaturing and reducing conditions, with addition of Laemmli buffer, blotted on a polyvinylidene difluoride membrane, and probed with anti-MMP-9 monoclonal antibodies at 1/1,000 final dilution. Bands were visualized by enhanced chemiluminescence. Densitometric analysis of the bands was performed using a computerized densitometer (Chemidoc).Assay of IL-1beta mRNA expression by real-time quantitative RT-PCRAfter termination of phagocytosis, monocytes were further incubated with Panserin 601 monocyte medium in a humidified CO2/air-incubator at 37°C for 6 h (immunopurified monocytes) or 15 h (adherent monocytes). Total cellular RNA from 2 × 106 cells was isolated from monocytes by TRIzol, according to the manufacturer's instructions, and eluted in 20 μl diethyl pyrocarbonate water. To remove any contaminating DNA, RNA was treated with Ambion's DNA-free kit (Ambion). Retrotranscription was performed using 6 μg of RNA, 200 U/μl of M-MLV and 25 μ/μl oligo-dT (Invitrogen). Real-time quantitative RT-PCR was performed with the iCycler instrument (Bio-Rad) and data analysis was performed with iCycler iQ Real-Time Detection System Software version 3.0 (Bio-Rad). IL-1beta (GenBank accession no. NM_000576) oligonucleotide sequences (forward: 5'-ACA GAT GAA GTG CTC CTT CCA-3', reverse: 5'-GTC GGA GAT TCG TAG CTG GAT-3') were identified using Beacon Designer Software package and designed to be intron-spanning allowing the differentiation between cDNA and DNA-derived PCR products. PCR amplification was carried out in 25 μl of reaction mixture. 1 μl of cDNA (corresponding to 105 cells) and 400 nM primers were added to the amplification mixture (iQ SYBR Green Supermix, Bio-Rad). DNA polymerase was pre-activated for 2 min at 94°C, and the amplification was performed by a 40-cycle PCR (94°C, 30 s, 60°C, 30 s and 72°C, 30 s). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as reference gene to normalize cDNA across samples. Relative quantitation for IL-1 beta, expressed as -fold variation over untreated control cells, was calculated using the 2-ΔΔCT method. To validate the use of the 2-ΔΔCT method, serial dilutions of cDNA from monocytes stimulated for 6 or 15 h by 20 ng/ml rhTNF, were tested. Analysed transcripts exhibited high linearity amplification plots (r > 0.98) and similar PCR efficiency (85.7% for IL-1 beta and 86.5% for GAPDH), confirming that the expression of each gene could be directly compared. The specificity of PCRs was confirmed by melt curve analysis. Values are means of triplicate measurements.ResultsEnhancement of IL-1beta production by adherent monocytes fed with HZ or trophozoitesAdherent monocytes were allowed to phagocytose HZ, trophozoites, nonparasitized opsonized RBCs and latex particles (control meals) during 3 h. As an average, each monocyte ingested 8–10 trophozoites, or HZ equivalent to 8–10 trophozoites in terms of ingested haem, or almost 8–10 non-parasitized anti-D IgG opsonized RBCs, as shown previously [1,19]. After termination of phagocytosis and elimination of noningested phagocytic meals by repeated washings, and further incubation during 48 h, IL-1beta production was measured by ELISA in cell supernatants. Compared to unfed control monocytes, IL-1beta production was increased approximately two-fold after phagocytosis of HZ or trophozoites, whereas phagocytosis of control meals did not affect significantly cytokine production (Figure 1). In selected experiments, adherent monocytes after the 3 h phagocytic period were further incubated for 48 h in presence of Merck's inhibitor I, a specific inhibitor of MMP-9/MMP-13 activity. Supplementation of this inhibitor did not affect cytokine production by HZ/trophozoite-fed monocytes.Figure 1HZ and trophozoite phagocytosis enhances IL1-beta production by human adherent monocytes. Human adherent monocytes were unfed or fed with HZ, trophozoites and control meals (IgG-anti D-opsonized nonparasitized RBCs, latex particles). After 3 h phagocytosis and a further incubation during 48 h, IL1-beta levels were measured by ELISA in cell supernatants. Data are given as ng IL-1beta/ml supernatant (mean values ± SD of six independent experiments). Data were analysed for significance by Student's t-test. Significance of differences (column numbers): trophozoite-fed(3)/HZ-fed(5) vs unfed-(1)/nonparasitized RBC-fed(2) monocytes, p < 0.05; latex-fed(4) vs unfed-(1)/nonparasitized RBC-fed(2) monocytes, n.s.; trophozoite-fed(3) vs HZ-fed(5) monocytes, n.s.Enhancement of MMP-9 protein expression and enzyme activity in adherent monocytes after HZ phagocytosis and rhIL-1beta treatment. Abrogation of the HZ effect by anti-hIL-1beta antibodiesUnfed, latex-fed and HZ-fed adherent monocytes after termination of phagocytosis were further incubated for 15 h (MMP-9 mRNA expression studies) or 48 h (MMP-9 activity and protein expression studies) in presence or absence of 20 ng/ml rhIL-1beta and 30 ng/ml blocking anti-hIL-1beta antibodies. Phagocytosis of HZ enhanced enzyme activity (measured in cell supernatants) and protein expression (measured in cell lysates), confirming previous data obtained by this group [8] (Figure 2, panel A). rhIL-1beta added to unfed or latex-fed monocytes mimicked the HZ effect at the protein expression/enzyme activity level (Figure 2, panel A and panel B) and at the mRNA expression level, and further enhanced enzyme activity when added to HZ-fed monocytes (Figure 2, panel A). Blocking anti-hIL-1beta antibodies abrogated the enhancement of protein expression (Figure 2, panel A) and enzyme activity (Figure 2, panel B), and also inhibited enhancement of mRNA expression observed after HZ phagocytosis.Figure 2HZ and trophozoite phagocytosis, and rhIL-1beta enhance MMP-9 protein expression (in cell lysates) and enzyme activity (in cell supernatants) in human adherent monocytes. Abrogation of the HZ effect by anti-hIL-1beta antibodies. Human adherent monocytes were unfed, fed with HZ or latex particles treated or not with rhIL-1beta (20 ng/ml) or blocking anti-hIL-1beta antibodies (30 ng/ml) as indicated. Panel A. Western blot with anti-MMP-9 antibodies and densitometric quantification of MMP-9 protein. After 3 h phagocytosis and a further incubation during 48 h, cell lysates were prepared, separated by PAGE (8% polyacrylamide) blotted and probed with anti-MMP-9 monoclonal antibodies (1/1000 final dilution). The 92-kDa band in the gel corresponds to pro-MMP-9. Data are given as arbitrary densitometric units (mean values ± SD of four independent experiments). Panel B. Gelatin zymography and densitometric quantification of MMP-9 enzyme activity. After 3 h phagocytosis and a further incubation during 48 h, cell supernatants were separated by PAGE (8% polyacrylamide gel containing 0.1% gelatin) under non-denaturing and non-reducing conditions. The 83-kDa negative bands in the gel correspond to MMP-9 enzyme activity. Data are given as arbitrary densitometric units (mean values ± SD of four independent experiments). Data (Panel A, Panel B) were analysed for significance by Student's t-test. Significance of differences (column/lane numbers): HZ-fed(7)/rhIL1beta(2)-stimulated vs control(1)/anti-hIL1beta-stimulated(3)/latex-fed(4) monocytes, p < 0.01 (Panel A) or p < 0.05 (Panel B). rhIL1beta-stimulated(2,5,8) vs HZ-fed(7,8) monocytes, n.s.; anti-hIL1beta-stimulated(3,6,9)/latex-fed(4) vs unfed(1) monocytes, n.s.Role of lipidic component of HZ in enhancement of IL-1beta production and MMP-9 activity in adherent monocytes after phagocytosis of HZPrevious work has shown that PUFAs stably adherent to the crystalline poly-haem core of native HZ are transformed by non-enzymatic haem catalysis into a number of potent lipoperoxidation derivatives [5]. To ascertain whether lipids were involved in HZ-elicited activation of MMP-9, lipid-free beta-haematin (synthetic HZ) and delipidized native HZ were fed to adherent monocytes. After phagocytosis, monocytes were further incubated for 48 hours and cell supernatants analysed by ELISA for IL-1beta production and MMP-9 activity. Beta-haematin and delipidized HZ were unable to enhance IL-1beta production (Figure 3, panel A) and stimulate MMP-9 activity (Figure 3, panel B).Figure 3IL-1beta production and MMP-9 enzyme activity (in cell supernatants) in human adherent monocytes unfed or fed with HZ, delipidized HZ or beta-haematin. Human adherent monocytes were unfed or fed with HZ, delipidized HZ (D-HZ) and beta-haematin. Panel A: IL-1beta production. After 3 h phagocytosis and a further incubation during 48 h, IL1-beta levels were measured by ELISA in cell supernatants. Data are given as ng IL-1beta/ml supernatant (mean values ± SD of four independent experiments). Data were analysed for significance by Student's t-test and differences between delipidized HZ or beta-haematin against unfed controls were not significant. Panel B: Gelatin zymography and densitometric quantification of MMP-9 enzyme activity. After 3 h phagocytosis and a further incubation during 48 h, cell supernatants were separated by PAGE and MMP-9 enzyme activity measured by gelatin zymography and densitometric quantification (see legend to Figure 2 for details). The 83-kDa negative band in the gel corresponds to MMP-9 enzyme activity. Data are given as arbitrary densitometric units (mean values ± SD of four independent experiments). Data (Panel A, panel B) were analysed for significance by Student's t-test. Significance of differences (column/lane numbers). HZ-fed(2) vs unfed(1)/D-HZ-(3)/beta-haematin(4)-fed monocytes, p < 0.01; unfed(1) vs D-HZ(2)/beta-haematin(4)-fed monocytes, n.s.Involvement of 15-HETE in HZ-mediated effects on IL-1beta production and MMP-9 activity in adherent monocytes and on IL-1beta mRNA expression in immunopurified monocytesPrevious work has indicated that 15-HETE, a product of arachidonic acid peroxidation by HZ, is an active mediator of HZ/trophozoite effects in monocytes [20]. As shown in Figure 4, panel A, 15-HETE added to adherent monocytes at 0.1–10 μM (final concentration) enhanced production of IL-1beta, measured in cell supernatants 48 h after addition. 15-HETE added in the same concentration range also stimulated MMP-9 activity, similarly measured in cell supernatants 48 h after addition (Figure 4, panel B). This second effect was not concentration-dependent. Both 15-HETE-mediated effects were comparable to those elicited by HZ/trophozoite phagocytosis. In selected experiments with immunopurified monocytes, IL-1beta mRNA expression measured 6 h after phagocytosis was increased 3,5-fold after HZ and 2-fold after addition of 10 μM 15-HETE (Figure 5). The stimulatory effect of 15-HETE appears to be specific, as 4-hydroxynonenal [4-HNE], another potent PUFA derivative generated by HZ activity [21] was unable to stimulate IL-beta production and MMP-9 activity when added at 0.1 μM (final concentration) and downregulated both parameters when added at 1–10 μM (final concentration).Figure 4IL-1beta production and MMP-9 enzyme activity (in cell supernatants) in human adherent monocytes unfed or fed with HZ and treated or not with 15-HETE. Human adherent monocytes were fed or not with HZ and treated or not with 15-HETE added at time 0 at 0.1–10 μM (final concentration). Panel A. After 3 h phagocytosis and a further incubation during 48 h (HZ-fed monocytes) or 48 h after addition of 15-HETE, IL-1beta levels were measured by ELISA in cell supernatants. Data are given as ng IL-1beta/ml supernatant (mean values ± SD of four independent experiments). Panel B. After 3 h phagocytosis and a further incubation during 48 h (HZ-fed monocytes) or 48 h after addition of 15-HETE, cell supernatants were separated by PAGE and MMP-9 enzyme activity measured by gelatin zymography and densitometric quantification (see legend to Figure 2 for details). The 83-kDa negative bands in the gel correspond to MMP-9 enzyme activity. Data are given as arbitrary densitometric units (mean values ± SD of four independent experiments). Data were analysed for significance by Student's t-test. Significance of differences (column/lane numbers). Unfed(1) vs HZ-fed(5) monocytes, p < 0.05; unfed(1) vs 15-HETE-treated (3,4,5) monocytes, p < 0.05 (Panel A) and p < 0.01 (Panel B).Figure 5IL-1beta mRNA expression in immunopurified human adherent monocytes unfed or fed with HZ and treated or not with 15-HETE. Human CD14+ immunopurified monocytes were unfed, fed with HZ or treated with 15-HETE added at time 0 at 1–10 μM (final concentration). 6 h after phagocytosis or addition of 15-HETE. mRNA expression was measured by real-time quantitative RT-PCR in cell lysates and expressed as -fold variation over untreated monocytes. Mean values ± SD of three independent experiments. Data were analysed for significance by Student's t-test. Significance of differences (column numbers). Unfed(1) vs HZ-fed(2) monocytes, p < 0.01; unfed(1) vs 15-HETE 1 μM(3) treated monocytes, n.s.; unfed(1) vs 15-HETE 10 μM(4) treated monocytes, p < 0.05.DiscussionMatrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes characterized by their ability to remodel/disrupt subendothelial matrix proteins and shed or activate cytokines from their precursors [9-12,22]. MMPs play physiological roles, for example in wound repair [22], and are also involved in pathological processes such as cancer metastasis [23] or neurological diseases [24]. Basal transcription level of MMPs is generally low [25], but it can be enhanced in various cell types including monocytes, by cytokines and growth factors, and by cell-cell or cell-matrix interactions [9]. Recently, involvement of MMPs in malaria has been described. Deininger et al [26] found higher levels of MMP-1 and angiogenic proteins such as VEGF in post-mortem samples of brain tissues of patients dead from cerebral malaria. Van den Steen et al [27] described higher MMP-9 expression in brain and other tissues of mice with cerebral malaria. The enzyme was apparently produced by cells of monocytic lineage. Lastly, present group [8] has shown that human adherent monocytes fed with HZ or HZ-containing trophozoites displayed increased activity and mRNA/protein expression of MMP-9, and increased production of TNF. Since TNF induces expression of MMP-9, while MMP-9 sheds TNF from its membrane-bound precursor, interaction between MMP-9 and TNF was considered to start a positive feedback loop eventually enhancing the pathological effects of both molecules: for MMP-9 – disruption of subendothelial basal lamina and infiltration of mononuclear cells in brain, lung and kidney – [28-30]; and for TNF, – fever, hypoglycaemia, circulatory failure, and placental pathology – [31-35].Present data show that IL-1beta production was enhanced in HZ/trophozoite-fed adherent monocytes, and causally related to enhancement of MMP-9 mRNA and protein expression (measured in cell lysates) and MMP-9 enzyme activity (measured in cell supernatants). Short-term experiments were performed to establish which cytokine (TNF or IL-1beta) was the primary target of HZ stimulatory activity. Data indicate that while blocking anti-hIL-1beta antibodies significantly reduced TNF production by HZ-fed cells at 1 h (p < 0.05) and at 2 h (p < 0.02), blocking anti-hTNF Abs did not affect the short-term production of IL-1beta by HZ-fed cells. These data seem thus to suggest that the enhancement of IL-1beta formation occurred first followed by enhanced formation and activity of TNF and MMP-9, respectively.HZ and HZ-containing trophozoites contain large amounts of monohydroxy derivatives of polyunsaturated fatty acids (OH-PUFAs). OH-PUFAs are stable derivatives of PUFA peroxidation, here most likely non-enzymatically generated by haem-catalyzed lipid peroxidation carried out by the poly-haem moiety of HZ [5]. High concentration of HZ and acidic conditions are likely to favour unspecific haem-catalyzed lipid peroxidation, leading to a complex pattern of oxygenated products. Six HETE isomers and two major isomers of HODE (hydroxyoctadeca-9Z,11E-dienoic acid, a linoleic acid derivative) were found in HZ and trophozoites [5]. Of those molecules and isomers, only 12- and 15-HETE mimicked toxic effects of HZ/trophozoite phagocytosis in monocytes, such as inhibition of oxidative burst and inhibition of differentiation and maturation of monocytes to dendritic cells [15]. HODE isomers were inactive. Native HZ was found to contain 0.24 mmole 15-HETE/mole haem. 15-HETE is further produced by ingested HZ, and HZ-fed monocytes were found to shed 15-HETE into the supernatant and to contain approximately 10 μM 15-HETE, under the realistic assumption of 10 RBC equivalents per monocyte and a monocyte volume of 500 fL [5]. 4-HNE, another PUFA derivative [21], supplemented here at 0.1 μM concentration did not stimulate IL-beta production or MMP-9 activity, while it downregulated both parameters at 1–10 μM concentration. Possible explanations may reside in the strong reactivity of 4-HNE with thiols, such as reduced glutathione, or amino-groups present in suspending buffers. Additionally, 4-HNE tends to concentrate in the cell membrane where it generates adducts with His and Cys residues of membrane proteins, possibly interfering with the 15-HETE transduction pathway.Based on present data, following sequence of events is likely. First, 15-HETE, possibly together with co-generated, similarly active 12-HETE, would induce production of IL-1beta and TNF via a yet undetermined transduction pathway. It is likely that the NF-kB pathway is involved in cytokine upregulation, as Jaramillo et al recently reported activation of the NF-κB pathway in HZ-fed murine macrophages [36,37]. Subsequently, increased IL-1beta and TNF would upregulate MMP-9 expression and activity. Indeed, literature data indicate the NF-KB pathway as essential for both TNF and IL-1beta induction, and the latter cytokines as potent upregulators of MMP-9 [38,39].ConclusionPhagocytosis of HZ or trophozoite-parasitized RBCs was shown to induce enhanced production of TNF and IL-beta, and to increase mRNA and protein expression (both measured in cell lysates), and enzymatic activity of MMP-9 (measured in cell supernatants), a metalloproteinase involved in disruption of basal membranes. Present data indicate that lipid components attached to HZ were instrumental for enhanced production of IL-1beta and MMP-9 increase. In fact, the ability of HZ to non-enzymatically generate HETEs and the presence of HETEs in HZ-fed monocytes [5], the lack of effects by feeding cells with delipidated HZ, and the recapitulation of HZ effects by supplementing exogeneous 15-HETE are converging indications that 15-HETE, an active member of the HETE family, may be causally involved in upregulation of IL1-beta and MMP-9. Thus, HZ-derived 15-HETE might be a molecule primarily responsible for cytokine induction and MMPs activation and possibly instrumental in inducing hallmarks of cerebral malaria such as localized haemorrhages and extravasation, migration and perivascular accumulation of phagocytic cells. Interestingly, it was recently shown that soluble factors released by trophozoite-parasitized RBCs significantly decreased electrical resistance of human brain-blood barrier endothelium, indicating a parasite-mediated perturbation of the brain monolayer barrier function [40].Authors' contributionsMP designed the research, performed the experiments and drafted the manuscript. VG performed the experiments and helped to draft the manuscript. GG helped with the real-time quantitative RT-PCR experiments. PA helped design the research, examined and interpreted the data and wrote the final manuscript. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2529305",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529305\nAUTHORS: Daniel Dodoo, Anastasia Aikins, Kwadwo Asamoah Kusi, Helena Lamptey, Ed Remarque, Paul Milligan, Samuel Bosomprah, Roma Chilengi, Yaa Difie Osei, Bartholomew Dicky Akanmori, Michael Theisen\n\nABSTRACT:\nBackgroundAntigen-specific antibody-mediated immune responses play an important role in natural protection against clinical malaria, but conflicting estimates of this association have emerged from immuno-epidemiological studies in different geographical settings. This study was aimed at assessing in a standardized manner the relationship between the antibody responses to four malaria vaccine candidate antigens and protection from clinical malaria, in a cohort of Ghanaian children.MethodsStandardized ELISA protocols were used to measure isotype and IgG subclass levels to Apical Membrane Antigen 1 (AMA1), Merozoite Surface Protein 1–19 (MSP119), Merozoite Surface Protein 3 (MSP3) and Glutamate Rich Protein (GLURP) antigens in plasma samples from 352 Ghanaian children, aged three to 10 years with subsequent malaria surveillance for nine months. This is one of a series of studies in different epidemiological settings using the same standardized ELISA protocols to permit comparisons of results from different laboratories.ResultsThe incidence rate of malaria was 0.35 episodes per child per year. Isotype and IgG subclasses for all antigens investigated increased with age, while the risk of malaria decreased with age. After adjusting for age, higher levels of IgG to GLURP, MSP119, MSP3 and IgM to MSP119, MSP3 and AMA1 were associated with decreased malaria incidence. Of the IgG subclasses, only IgG1 to MSP119 was associated with reduced incidence of clinical malaria. A previous study in the same location failed to find an association of antibodies to MSP119 with clinical malaria. The disagreement may be due to differences in reagents, ELISA and analytical procedures used in the two studies. When IgG, IgM and IgG subclass levels for all four antigens were included in a combined model, only IgG1 [(0.80 (0.67–0.97), p = 0.018)] and IgM [(0.48 (0.32–0.72), p < 0.001)] to MSP119 were independently associated with protection from malaria.ConclusionUsing standardized procedures, the study has confirmed the importance of antibodies to MSP119 in reducing the risk of clinical malaria in Ghanaian children, thus substantiating its potential as a malaria vaccine candidate.\n\nBODY:\nBackgroundMalaria remains one of the most important causes of morbidity and mortality in the world. Current methods of control are only partially effective and, therefore, the development of a vaccine which can provide a high degree of protection is a priority. Antibody-mediated immune responses to malaria antigens are known to be involved in protecting against disease [1-4], but the antigens that induce protective antibodies have not been conclusively identified. Immuno-epidemiological studies from different laboratories have sometimes yielded conflicting results [5-8]. This may be partly due to differences in malaria endemicity and the use of different study designs, reagents, assay protocols and statistical methodologies. In an attempt to make such studies more comparable, the Afro-Immuno Assay (AIA) network project was initiated. The network includes six African Institutions in Gabon, Ghana, Burkina Faso, Senegal, Tanzania, and Zimbabwe and three European Institutions from Denmark, The Netherlands and France. The Afro-Immuno Assay network has developed standardized enzyme immuno assays [9-11] that ensure the use of the same reagents, protocols and statistical methods to assess the association between acquisition of malaria specific antibody responses to four potential malaria vaccine candidate antigens and possible protection from clinical malaria. Samples for the AIA multi-center project were retrospectively obtained from cohort studies in six different geographical and epidemiological settings, comprising low endemic to holoendemic areas. These antigens include the Glutamate Rich Protein (GLURP), the Merozoite Surface Protein 3 (MSP3) [12], the 19-kilo Dalton region of the Merozoite Surface Protein 1 (MSP119) [13] and the Apical Membrane Antigen 1 (AMA1) [14], which are all thought to induce protective antibody responses through various mechanisms [15-18]. Vaccines incorporating these antigens are currently in clinical trials and are described in detail elsewhere [7,19-26]. It is likely that a future malaria vaccine will comprise multiple rather than single antigens and it is, therefore, useful to study natural immune responses to multiple malaria antigens in relation to incidence of malaria in a more standardized way. In this study, the standardized AIA ELISA procedures [9-11], were used to assess the relationship between incidence of clinical malaria and naturally acquired isotype and IgG subclass responses to these four leading malaria vaccine candidate antigens, AMA1, MSP119, MSP3 and GLURP in Ghanaian children from three to 10 years of age.Materials and methodsStudy area, study population and morbidity surveillanceSamples used in this study were obtained in March 2002 from a longitudinal study conducted in Dodowa, in which 352 children aged three to 10 years (in the active phase of acquiring immunity to malaria), were enrolled in a study, whose original aim had been to assess the role of cytokine regulation and immunity to malaria. Dodowa is a semi-rural town in the Dangme West District of the Greater Accra Region of Ghana, about 50 km from the capital Accra and is an area of moderate and stable malaria transmission with a seasonal peak. Bed net coverage in this area was low, about 10% [27]. The study was approved by the Noguchi Memorial Institute's Ethical Review Board. After obtaining consent from parents, blood was obtained from each child and plasma stored at -20°C until use. Malaria episodes were detected using both active and passive surveillance implemented over a period of nine-months, spanning the entire malaria transmission season. Clinical and parasitological information was captured using a standard questionnaire. Each child was visited once a week and the child and the parents or guardians were asked about symptoms of malaria since the last visit and whether she/he had received any anti-malarial treatment. The child was then given a physical examination and the body temperature measured. Children with a history of fever within 48 hours and/or axillary temperature equal to or above 37.5°C had a rapid test for malaria parasitaemia using OptiMAL™ (DiaMed, FLOW Inc. Portland, Oregon) and then thick and thin blood films were prepared for microscopy for estimating the parasite density which was used later in the more specific malaria case definition employed for data analysis. Only children with measured or reported fever, and with a positive rapid test were treated with chloroquine (in accordance with the then prevailing national malaria treatment policy) and in the case of severe symptoms, the child was referred to the hospital. At monthly intervals, blood smears from finger pricks were obtained from all children irrespective of symptoms to estimate the prevalence of asymptomatic malaria infections. The parents of the children were instructed to report to the field team if the child had any symptoms of disease at any time. The qualifying case definition for malaria in the data analysis was reported fever and/or a measured temperature equal to or above 37.5°C, with parasitaemia ≥ 5,000 parasites/μl of blood; this case definition has been found to have 90% sensitivity and specificity in the study area [28-30]. Positive and negative control plasma used in ELISA measurements were obtained from adult Liberians and Danes respectively.Malaria antigensAMA1 was from the Pichia pastoris expressed ectodomain of Plasmodium falciparum FVO strain comprising amino acids 25–545 [31] (Donated by A Thomas, Biomedical Primate Research Centre). GLURP was an Escherichia coli recombinant protein containing the conserved non-repeat N-terminal region (amino acids 25–514) called R0 [32] (Donated by M. Theisen, Statens Serum Institut). MSP119 was a Baculovirus antigen of the C-terminal region of the merozoite protein surface 1, produced in insect cells infected with a recombinant Baculovirus containing a synthetic G-C enriched PfMSP1 gene (Palo Alto allele), coding for 43 N-terminal MSP1 precursor residues and 16 amino acid residues upstream of the \"classical\" MSP-119 (NIS---FCS) [33] (Donated by S. Longacre, Institut Pasteur). The MSP3 antigen used in this study was a long synthetic peptide called LR55 (amino acids 181 – 276) of the merozoite surface protein 3 [34] (Donated by M. Theisen, Statens Serum Institut). All the antigens were provided through the AIA Project.Enzyme-linked immunosorbent assay (ELISA)Specific isotype and IgG subclass levels against GLURP, MSP119, MSP3 and AMA1 were measured using indirect ELISA according to the AIA standard ELISA protocols [9-11]. All antigens tested were optimized and shown to be stable for at least three weeks, when antigen-coated plates and serum/plasma dilutions are refrigerated. The subclass specific reagents used were selected on the basis of low cross reactivities among themselves. To control for inter-assay and day-to-day variations in the standardized ELISA procedure, three-fold serial dilutions of reference standard reagents (IgG, IgM and IgG1 to IgG4) were directly coated on each ELISA plate (Maxisorp Nunc, Denmark) at a start concentration of 1,000 ng/ml (100 μl/well). OD values for the test samples were converted into antibody units with the standard reference curves generated for each ELISA plate using a four parameter curve-fit Microsoft Excel-based application. Samples were re-tested if the coefficient of variation between duplicate absorbance values were higher than 15% and plates were also re-tested if the R-square value of the standard curve was less than 97%. The reference standards, PBS buffer blank, positive and negative control plasma pools that were included in each ELISA test plate allow for the determination of failed assay runs. The AIA ELISA procedure used in this study is described in detail elsewhere [10].Statistical analysisClinical data were double entered using Microsoft Fox Pro and immunological data using Excel. STATA version 9.2 (Statcorp, Texas) was used for statistical analysis. Children were considered to have a clinical malaria episode if they had parasitaemia of ≥ 5,000 parasites/μl, with a measured temperature ≥ 37.5°C or a history of fever in the last 48 hours [6,35,36].For each antigen, Poisson regression was used to investigate the association between the levels of antibody measured at baseline and the incidence rate of the first (or only) episode of clinical malaria. The level of total IgG, IgM and each IgG subclass, were analysed for each antigen in turn. Antibody values were transformed to log base 2, so that the rate ratio represents the ratio of malaria incidence corresponding to a doubling of antibody level. To investigate whether the relationship between malaria incidence and antibody level was nonlinear, a likelihood ratio test was used to compare the fit of the model when antibody level was included as a categorical or a continuous variable. When there were zero antibody values, indicating levels below the detection limit, the zero (left censored) values were assigned a nominal value equal to half the smallest measured value for that variable. If the proportion of zero values was large, the variable was treated as categorical with the reference category containing the zero values and the positive values divided into three equal groups. A likelihood ratio test was used to determine the P-value for the association with malaria incidence. Age at enrolment was considered to be an important potential confounder, and was included in the regressions as a factor with categories defined by quintiles. To model seasonality in malaria incidence, the calendar month of surveillance was included in the models as a factor. To construct a parsimonious model using all the immunological variables, firstly a model was produced for each antigen; in this model each IgG subclass, total IgG and IgM were candidates for inclusion provided the P-value for association with malaria incidence was 0.1 or less when considered individually. Variables were then removed from the model if the P-value for the likelihood ratio test was more than 0.1, provided removal did not change coefficients of variables in the model by more than 10%. In a second stage, the variables included in these models were candidates for inclusion in a final model derived in a similar way. Baseline parasitaemia was not considered as a potential confounder, but the interaction between each immunological variable and the presence of parasitaemia at baseline in their effects on malaria incidence was examined. A consequence of using a more specific case definition in the analysis than was used to decide treatment during the study is that children could have received anti-malarial treatments during the period they are considered at risk in the analysis. To explore the impact of these drug treatments, a time dependent variable was defined, to allow for a reduced risk of malaria for a period of 28 days after each drug treatment, which was included as a covariate in the Poisson regression model. LOESS smoothing was applied in R software to plot antibody levels in relation to age. Spearman's rank correlation test was used to assess associations between antibody levels and age.ResultsPattern of P. falciparum infections and malaria in the study cohortOf the 352 children recruited for the original study, eight were lost to follow-up immediately after the baseline blood sampling. Of the 344 children followed up, sixty four (19%) had at least one episode of malaria (53 children had one episode, nine had two and two had three episodes). The incidence rate of malaria in the study cohort was 0.35 attacks per child per year (Table 1). The risk of clinical malaria decreased with increasing age [37]. Sixty-six percent of the children had asymptomatic parasitaemia at baseline. Parasites were predominantly P. falciparum (95%) and the prevalence of parasitaemia measured each month was roughly constant (ranging from 50% to 65%). The incidence of clinical malaria, however, varied during the survey period, rising gradually from March to May, peaking in July and then decreasing until November (Figure 1).Table 1Malaria incidence by age groupAge in yrs:No. of childrenCum. incidence of malariano episodes of malaria (years at risk)Incidence rate per child year (95%CI)33212/32 (38%)15 (19.52)0.79 (0.48–1.31)4–58624/86 (28%)28 (54.75)0.51 (0.35–0.74)6–77911/79 (14%)13 (51.38)0.25 (0.15–0.44)8486/48 (13%)7 (31.11)0.22 (0.11–0.47)9–109911/99 (11%)14 (61.22)0.23 (0.14–0.39)Total34464/344 (19%)77 (217.48)0.35 (0.28–0.44)Figure 1The monthly point prevalence of asymptomatic malaria parasitaemia and incident rate of clinical malaria. The point prevalence of asymptomatic parasitaemia for each month is represented as a bar graph and the pattern of clinical malaria (incident rate) is shown as a line graph.Relationship between age and antibody levelsSeveral studies in malaria endemic regions have shown increasing antibody levels with age. This pattern is more pronounced the greater the intensity and duration of malaria transmission. In this study, the relationship between antibody levels and age were assessed for antibodies against MSP119, AMA1, GLURP and MSP3. The levels of IgG and IgM to MSP119, MSP3, AMA1 and GLURP increased with age (Spearman correlation coefficient (rs) 0.21 – 0.45; p < 0.001, Figure 2). IgG levels to AMA1 however, gradually increased until six years of age, and then leveled off. For the four antigens tested, IgG1, IgG2 and IgG3 significantly increased with age (rs, 0.12 – 0.36; p < 0.03) with the exception of IgG2 to AMA1. There was no evidence that the level of IgG4 was associated with age for any of the antigens. Like IgG to AMA1, IgG3 to MSP3 steadily increased with age until seven years of age, then leveled off (Figure 3).Figure 2Baseline IgG and IgM levels in relation to age. The top and bottom panels represents total IgG or IgM levels to AMA1, GLURP, MSP119 and MSP3 in relation to age of Ghanaian children, respectively. The line shows the LOESS smoothed estimate of the geometric mean.Figure 3Baseline IgG subclass levels in relation to age. The top, 2nd, 3rd and 4th panels represent IgG subclasses 1 to 4 against AMA1, GLURP, MSP119 and MSP3 in relation to age of Ghanaian children, respectively. The line shows the LOESS smoothed estimate of the geometric mean.The levels of both IgG and IgG1 were highest for AMA1 and lowest for MSP3, whereas IgG levels to MSP119 and GLURP were comparable (Figures 2 and 3). The levels of IgG3 were higher in AMA1 than the comparable levels in GLURP, MSP119 and MSP3 (Figure 3). In general, the levels of cytophilic IgG1 and IgG3 were higher than those of non-cytophilic IgG2 and IgG4, IgG4 levels being the lowest.Antibody levels in relation to protection from clinical malariaTotal IgG to MSP3, MSP119, and GLURP, and IgM to all four antigens tested (MSP3, MSP119, GLURP, and AMA1) were associated with reduced malaria incidence in crude analyses (Table 2). The incidence of both clinical malaria and antibody levels were associated with age, age is therefore a potential confounder, and it is important to adjust for its effects. After adjusting for the effect of age, there was evidence of a significant association between total IgG to MSP3, MSP119, GLURP and reduced risk of malaria (IgG to MSP3: rate ratio 0.69 (95%CI 0.53, 0.90) P = 0.01; IgG to MSP119: 0.75 (0.61, 0.92) P = 0.01; IgG to GLURP: 0.79 (0.64, 0.98) P = 0.04); and IgM levels to AMA1, MSP3, MSP119 were also significantly associated with reduced risk of malaria (Table 2).Table 2Association of total IgG and IgM with malaria incidenceIgGCrude IRR (95%CI)P-valueIRR adjusted for age (95%CI)P-valueMSP30.59 (0.45, 0.76)< 0.00010.69 (0.53, 0.90)0.01MSP1190.74 (0.63, 0.88)< 0.00010.75 (0.61, 0.92)0.01GLURP0.68 (0.55, 0.82)< 0.00010.79 (0.64, 0.98)0.04AMA10.96 (0.84, 1.09)0.501.03 (0.89, 1.20)0.67IgM:MSP30.62 (0.49, 0.80)< 0.00010.76 (0.59, 0.97)0.03MSP10.59 (0.47, 0.75)< 0.00010.68 (0.53, 0.88)< 0.01GLURP0.71 (0.58, 0.87)< 0.010.84 (0.68, 1.03)0.09AMA10.48 (0.34, 0.67)< 0.00010.63 (0.44, 0.91)0.01Malaria incidence rate ratios (IRR) with 95% confidence interval, corresponding to a doubling of baseline antibody level, before and after adjustment for the effect of age.A large proportion of the measurements of IgG4 to MSP3 and AMA1 and IgG2 to AMA1 were zero (left-censored) values and so these variables were treated as categorical variables (Table 3). In the crude analysis, IgG1, IgG2 and IgG3 to MSP3, MSP119, GLURP and also IgG4 to MSP3, were associated with a reduced risk of clinical malaria (Table 3, 4), but after adjustment for age, only one of these variables, IgG1 to MSP119, remained significantly associated with malaria incidence (rate ratio 0.89 (95%CI 0.80, 0.99), P = 0.04, Table 4).Table 3Association of IgG2 and IgG4 with malaria incidence.AntigenAntibodyCrude IRR (95%CI)P-valueAge-adjusted IRRP-valueMSP3IgG2*(coded as log(2))0.74 (0.57, 0.98)0.040.85 (0.63, 1.15)0.29IgG4< detection limit10.0110.20< median0.45 (0.21, 0.95)0.56 (0.26, 1.19)≥ median0.47 (0.28, 0.79)0.68 (0.40, 1.15)MSP119IgG2*(coded as log(2))0.62 (0.45, 0.86)< 0.010.836 (0.606, 1.157)0.275IgG4*(coded as log(2))1.07 (0.95, 1.21)0.250.838 (0.608, 1.159)0.278GLURPIgG2*(coded as log(2))0.65 (0.49, 0.86)< 0.010.79 (0.58, 1.08)0.13IgG4*(coded as log(2))0.94 (0.81, 1.08)0.371.01 (0.87, 1.16)0.94AMA1IgG2< detection limit10.1810.54< median1.80 (0.86, 3.78)≥ median1.78 (0.90, 3.51)IgG4< detection limit10.1910.54< median0.96 (0.47, 1.93)≥ median0.62 (0.36, 1.07)For IgG 2 to AMA1 and for IgG4 to AMA1 and MSP3, antibody level was categorized.*For IgG2 to MSP3, MSP119 and GLURP, the association is expressed as the incidence rate ratio (IRR) corresponding to a doubling of baseline antibody level.Table 4Association of IgG1 and IgG3 with malaria incidenceIgG1:Crude IRR (95%CI)P-valueIRR adjusted for age (95%CI)P-value MSP30.82 (0.72, 0.94)< 0.010.88 (0.76, 1.03)0.11 MSP1190.86 (0.77, 0.97)0.010.89 (0.80, 0.99)0.04 GLURP0.85 (0.74, 0.98)0.020.93 (0.81, 1.08)0.35 AMA10.95 (0.86, 1.06)0.361.01 (0.90, 1.14)0.83IgG3: MSP30.90 (0.82, 0.98)0.010.94 (0.86, 1.03)0.17 MSP1190.90 (0.82, 0.99)0.030.93 (0.84, 1.03)0.18 GLURP0.83 (0.74, 0.93)< 0.010.91 (0.80,1.03)0.14 AMA10.97 (0.89, 1.06)0.480.98 (0.90, 1.07)0.73The incidence rate ratio (IRR) corresponds to a doubling of baseline antibody level, before and after adjustment for effects of ageWhen all immunological variables were considered simultaneously, only two variables were independently associated with reduced malaria incidence, IgG1 [(0.80 (0.66–0.96), p = 0.018)] to MSP119, and IgM [(0.48 (0.32–0.72), p < 0.001)] to MSP119 (Table 5).Table 5Adjusted rate ratios for immunological variables independently associated with malaria risk in the final model.Crude IRR (95%CI)P-valueIRR adjusted for age (95%CI)P-valueIRR adjusted for effects of age and treatment (95%CI)IgM to MSP1190.44 (0.30–0.64)P < 0.0010.49 (0.33–0.73)P < 0.0010.48 (0.32–0.72)IgG1 to MSP1190.78 (0.65–0.93)P = 0.0050.80 (0.66–0.96)P = 0.0180.80 (0.67–0.97)Treatment effect0.28 (0.07–1.15)P = 0.076--0.25 (0.06–1.05)The estimated rate ratio for the effect of antimalarial treatments given to children with parasitaemia < 5000/uL was 0.25 (95%CI 0.06–1.05), indicating these children had a substantially reduced risk of being found positive with parasitaemia >= 5000/ul during the 28 days following the treatment. But the estimates of rate ratios for other variables in the model were unchanged when this variable was included in the model suggesting treatment effects did not cause a bias in the estimation of the effects of immunological variables.DiscussionThis study in Ghanaian children is one of a series of studies designed to assess, using standardized methods, the association of antibody levels to four leading asexual blood-stage malaria antigens (MSP119, MSP3, AMA1 and GLURP) with the incidence of clinical malaria in different epidemiological settings. Previous results have been difficult to interpret due to different study protocols and analytical methods having been used [5-7,9,14,29,38-40]. In this study, the prevalence of asymptomatic malaria parasitaemia was relatively high and stable, while incidence of clinical malaria fluctuated in parallel with the intensity of transmission and seasonal rainfall pattern. These patterns are typical of this area and have been reported in previous studies [6,41-43]. The variation in the incidence of clinical malaria during the study period may be due to the introduction of new parasites with different antigenic presentation into the population leading to clinical malaria in susceptible individuals. The risk of malaria decreased with age, while isotype and IgG subclass levels to the four antigens generally increased with age. This is consistent with the hypothesis that immunity to malaria is largely effected through antibody-mediated mechanisms and that protective antibody levels to relevant antigens increase with age-related exposure to the parasites [44]. Increasing IgG and IgM levels with age may reflect greater cumulative exposure of older children but may also be due to older children having a more mature immune system [45]. The association of IgM responses with reduced malaria incidence indicates a possible role in immunity in Ghanaian children. Although much emphasis has been placed on IgG as the important isotype in immunity against malaria, IgM, which has lower affinity but is multivalent, may afford protection via other mechanisms such as the blocking of merozoite invasion of erythrocytes, complement activation, agglutination of merozoites [46].The association with malaria incidence of IgG responses to MSP3, MSP119 and GLURP is consistent with data from several other immuno-epidemiological studies [5,7-9,12,29,30,38,47-49] indicating that these antigens may be targets of protective antibodies [1,2,4]. There was no evidence that IgG levels to AMA1 were associated with malaria incidence, and there was no evidence of an interaction with baseline parasitaemia in contrast with similar studies conducted in Kenya [14]. As shown in other studies, the cytophilic antibody levels to the four antigens tested in this study were higher than the non-cytophilic ones, emphasizing their importance in anti-malaria immunity [9,29,38,50,51]. IgG and cytophilic antibody levels were highest for AMA1, while the levels were relatively low for MSP3. These differences in specific antibody levels may be related to the number of immunogenic B-Cell epitopes exposed to the immune system and could also be related to the structure, location and function of the particular antigen(s). With the exception of IgG2 levels to AMA1, IgG2 levels to GLURP, MSP119 and MSP3 increased with age which may suggest that IgG2 is involved in immunity against malaria. In recent studies, malaria antigen specific IgG2 have been shown to bind with high affinity to mutant Fcγ RII H131->R receptors [52] on monocytes, granulocytes and B cells, thus affording protection against malaria through monocytes and or neutrophil mediated mechanisms in subjects expressing the mutant CD32 form [53]. There was however, no evidence found that IgG2 was associated with malaria incidence for any of the four antigens tested.The IgG subclasses, IgG1, IgG2 and IgG3 for MSP3, MSP119, GLURP and IgG4 to MSP3 were associated with a reduced risk of malaria in un-adjusted analysis but of these only IgG1 to MSP119 was independently associated with malaria incidence after adjustment for age. Other studies have shown the importance of IgG1 in clearing parasitaemia in children [5,47,54] In a previous cohort study conducted in the same area, there was no association between antibody levels to MSP119 and malaria incidence [6]. This may be due to differences in antigen and antibody reagents used in the two studies; the MSP119 used in this study was a Baculovirus product that included a synthetic G-C enriched PfMSP1 gene that coded for the 43 N-terminal MSP1 precursor residues and 16 amino acid residues upstream of the \"classical\" MSP-119 (NIS---FCS) [33] compared to the one produced in E. coli, which had been used in the previous study. Although antibodies to MSP119 have been shown to be associated with both exposure and protection from disease, the fine specificities of such responses may contribute to protection [40]. The antigen used in this study may have assessed antibodies of fine specificities that are protective [40,55,56], whereas the antigen used in the previous study did not. It may, therefore, be important to assess in a standardized way the various MSP119, and other antigens, that are produced in different expression systems, in order to select the most appropriate antigen/expression system for malaria vaccine development. In another study in this series, in Burkina Faso, the same antigens, reagents, ELISA procedure and analytical methods were used; none of the isotypes and subclasses to MSP119 was associated with the incidence of clinical malaria. Since the same laboratory methods were used, the different outcomes of these two studies may be attributed to differences in malaria transmission or to the age of the children [44]. In Burkina Faso, the malaria transmission season is much shorter, which may influence the induction of differing antibody types for controlling malaria as shown in recent studies conducted in areas with different malaria endemicities in Tanzania [9,44]. Although total IgG to GLURP and MSP3 were associated with the risk of malaria, none of the constituent subclasses was identified to be associated with protection. When the effects of all the immunological variables were considered simultaneously, only IgG1 and IgM to MSP119 were independently associated with the incidence of clinical malaria, which may indicate the importance of MSP119 in malaria vaccine development. Parasite growth inhibition assays would be required to confirm if this association reflects a functional role of MSP119 in immunity.ConclusionIn conclusion, using standardized AIA ELISA, anti-MSP19 antibodies (IgG1 and IgM) have been shown to be the most strongly correlated with reduced risk of clinical malaria among the four malaria vaccine candidates tested. The standardized AIA ELISA developed for this project could be used to validate malaria vaccine candidate antigens, provide useful baseline information for clinical trials, and contribute to quality assured laboratory capacity in Africa.Authors' contributionsDD carried out field studies, developed assays and drafted the manuscript. AA performed the ELISA, compiled data and assisted in the manuscript writing. KAK and HL assisted with the ELISA. MT assisted with the assay development and manuscript writing, while PM, SB and ER wrote the analysis plan, performed the data analysis and together with RC, BDA and YDO contributed to the writing of the manuscript. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
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batch_10/PMC2529468.json

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+{
+    "id": "PMC2529468",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529468\nAUTHORS: Yuhong Nie, Dongmei Cui, Zhujuan Pan, Jiangyun Deng, Qiang Huang, Kaili Wu\n\nABSTRACT:\nPurposeThe present study was undertaken to investigate whether transforming growth factor-β (TGF-β) isoforms (TGF-β1, TGF-β2, and TGF-β3) and SMADs (SMAD2 and SMAD3) are involved in herpes simplex virus type 1 (HSV-1) corneal infection.MethodsHuman corneal epithelial cells (HCE) were infected with HSV-1 at a multiplicity of infection of 5. Cell morphological changes were observed under phase-contrast microscopy. Levels of mRNA for TGF-β isoforms 1, 2, and 3 as well as for SMAD2 and SMAD3 were measured by reverse transcription polymerase chain reaction (RT–PCR) at 0 h, 4 h, 8 h, 12 h, and 24 h after infection. Protein expression of TGF-β1, TGF-β2, SMAD3, and phospho-SMAD3 were analyzed by indirect immunofluorescence at 0 h, 12 h, and 24 h post-infection (p.i.) in HCE cells. Protein expression of TGF-β1 was also evaluated by ELISA.ResultsFollowing HSV-1 infection, a cytopathic effect in HCE cells was observed at 8 h p.i. and became significant at 24 h p.i. Compared with normal cells, the mRNA levels of TGF-β1 in HSV-1 infected HCE cells decreased significantly at 8 h, 12 h, and 24 h p.i. (p<0.01), and the expression of SMAD3 was also dramatically decreased 12 h and 24 h p.i. (p<0.01). No noticeable changes were found as a result of infection with respect to levels of TGF-β2, TGF-β3, and SMAD2 in HCE cells. Protein expression of TGF-β1, SMAD3, and phospho-SMAD3 decreased in infected cells at 12 h and 24 h p.i. compared with normal cells, but TGF-β2 had no change.ConclusionsTGF-β1 and SMAD3 may be involved in the pathology of corneal diseases associated with HSV-1 infection.\n\nBODY:\nIntroductionHerpes simplex virus type 1 (HSV-1) is a large, enveloped, double-stranded DNA virus with a genome of approximately 150 kbp. HSV-1 is widespread in the human population and commonly causes infections of the skin or mucosal surfaces. Occasionally, it can cause serious diseases such as sporadic encephalitis and ocular infections [1,2]. In the eye, HSV-1 infection usually results in blepharitis, conjunctivitis, corneal epithelial keratitis, and ulcerative and/or stromal keratitis [3]. The pathologies of these diseases are associated with several events such as the infiltration of neutrophils and mononuclear lymphocytes and the expression of growth factors, proinflammatory factors, and cytokines, which include transforming growth factor-β (TGF-β), IL-2, IL-6, IL-8, TNF-α, and interferon-β (IFN-β) [4-6]. These studies suggest that growth factors and cytokines are extremely important in regulating the body’s reaction to viral infection.TGF-β is a superfamily of cytokines, which affect a range of biological processes such as cell growth, differentiation, matrix production, migration, and apoptosis [7]. Furthermore, the TGF-β pathway is an important target for several viral proteins that interfere with signal transduction and transcription control in infected cells [8-11]. Upon activation of the TGF-β signaling pathway, TGF-β family members bind to the TGF-β type II receptor (TβR-II). TβR-II then recruits and phosphorylates TGF-β type I receptors (TβR-I), which in turn phosphorylates the intracellular effectors (i.e., SMAD2 and SMAD3). Subsequently, SMAD2 and SMAD3 complexes, which are associated with SMAD4, are translocated into the nucleus and regulate the transcription of target genes [7,12,13]. A previous study demonstrated that TGF-β isoforms are expressed in the human cornea [14,15], and TGF-β is believed to be one of the major factors involved in cell migration in the cornea and corneal wound healing [16-18]. Furthermore, TGF-β signaling through the SMAD pathway is activated in response to corneal wounds in which the basement membrane is removed [16]. Earlier studies suggested that TGF-β might be important in the pathology of various disease processes involved with viral infection. This has been demonstrated for a variety of viruses including cytomegalovirus (CMV), human immunodeficiency virus (HIV), canine distemper virus, rhinovirus, HSV-1, and human T-cell leukemia virus (HTLV) [8,9,19-22]. Corneal epithelial cells are the first line of defense against microbial infection and against further damage to the underlying stroma. Therefore, we must understand the role of TGF-β in the pathology of viral infection in the corneal epithelium. It is reasonable to suppose that TGF-β and SMADs play a critical role in the pathology of HSV-1 infection in the cornea. The present study was undertaken to examine whether TGF-β isoforms and SMADs (SMAD2 and SMAD3) are involved in HSV-1 corneal epithelial infection in vitro.MethodsCell cultureThe human corneal epithelial cell line (HCEC) that we used was described previously [23]. Cells were cultured in DMEM/high glucose supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 10 ng/ml human epidermal growth factor (EGF; Sigma, St Louis, MO), 5 μg/ml insulin, 5 μg/ml human transferrin (Sigma), and 0.4 μg/ml hydrocortisone (Gibco BRL, Grand Island, NY). The cells were incubated at 37 °C in a 5% CO2-95% air incubator. Experiments were performed when cells were at 80%-90% confluence.Virus infectionStocks of the HSV-1 (F strain) used in this study were propagated on HEp-2 cells grown in DMEM/F12, which contained 10% newborn bovine serum. The titer of virus stocks was determined according to a previously described method [24]. After cells were grown to 80%-90% confluence, cells were infected at a multiplicity of infection (MOI) of 5. After 1 h of adsorption at 37 °C with intermittent rocking, the inoculum was removed, and the medium was replaced with serum-free DMEM/high glucose. At the indicated times, cells were harvested for further experiments. To confirm virus infection, two virus genes (i.e., DNA polymerase and VP16) of HSV-1 were examined by reverse transcription polymerase chain reaction (RT–PCR) using the primers listed in Table 1. Two genes were detected in HSV-1 infected cells, which implied that HCE cells were successfully infected by HSV-1.Table 1Primer sequences and length of amplicons.GenePrimer sequencesProduct size (bp)TGF-β1forward: 5′-GGGACTATCCACCTGCAAGA-3′239 reverse: 5′-CCTCCTTGGCGTAGTAGTCG-3′TGF-β2forward: 5′-GTGGAGGTGCCATCAATA-3′499 reverse: 5′-GAGGAGCGACGAAGAGTA-3′TGF-β3forward: 5′-CAA AGGGCTCTGGTGGTC-3′216 reverse: 5′-CGGGTGCTGTTGTAAAGTG-3′SMAD3forward: 5′-AGGAGAAATGGTGCGAGA A-3′197 reverse: 5′-CCACAGGCGGCAGTAGAT-3′SMAD2forward: 5′-TCACAGTCATCATGAACTCAAGG-3′471 reverse: 5′-TGTGACGCATGGAAGGTCTCTC-3′DNA polymeraseforward: 5′-ATCAACTTCGACTGGCCCTT-3′179 reverse: 5′-CCGTACATGTCGATGTTCAC-3′VP16forward: 5′-GGTCGCAACAGAGGCAGTCA-3′418 reverse: 5′-CCCGAACGCACCCAAATC-3′GAPDHforward: 5′-GCACCGTCAAGGCTGAGAAC-3′138 reverse: 5′- TGGTGAAGACGCCAGTGGA-3′RNA isolation and reverse transcription polymerase chain reaction analysisCells were harvested and washed with phosphate buffered saline (PBS). Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The quantity and quality of total RNA were estimated by spectrophotometry and agarose electrophoresis. Subsequently, RNA was reverse-transcribed into cDNA using a RevertAidTM First Strand cDNA synthesis kit (Fermentas, Glen Burnie, MD). cDNA was then amplified by GoTaq® Green Master mix (Promega, Madison, WI) using the specific primers listed in Table 1. The PCR products were electrophoresed in GoldViewTM stained 2% agarose gels (SBS Genetech, Beijing, China). Quantification of the bands was performed using a BioImaging System (UVP, Upland, CA) and Gel-pro software (Media Cybernetics, Bethesda, MD), and the level of mRNA was expressed as the ratio of integrated optical density (IOD) of specific PCR products over GAPDH IOD.Indirect immunofluorescenceHCE cells were cultured on a glass coverslip in 12 well chamber dishes and infected with HSV-1 as described above. At the indicated times, changes in cellular morphology were photographed using a phase-contrast microscope. Slide-mounted cells were used for indirect immunofluorescence analysis according to the method described previously [25]. The cells were blocked by endogenous peroxidase-blocking solution and followed by goat serum (each for 10 min at 37 °C). After blocking nonspecific binding, cells were incubated with rabbit anti-human monoclonal/polyclonal antibodies that recognize TGF-β1 (Santa Cruz, Delaware Avenue, CA), TGF-β2 (Santa Cruz), SMAD3, and phospho-SMAD3 (both from Cell Signaling, Danvers, MA) at 4 °C overnight. Cells were then incubated with FITC-conjugated secondary goat anti-rabbit IgG (Zhongshan Goldenbridge, Beijing, China) at 37 °C for 1 h. Prior to mounting, cells were stained with propidium iodide (PI) for 10 min. Cells were then observed using a confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Cells incubated with PBS (instead of the first antibody) were used as negative controls.Measurement of TGF-β1 protein in human corneal epithelial cells by ELISAAt 0 h, 12 h, and 24 h p.i., HSV-1 infected HCE cells were lysed with lysate buffer (Pierce, Rockford, IL). The samples were sonicated and centrifuged at 12,000 rpm for 30 min at 4 °C to remove cellular debris. Protein content in the supernatant was determined by the bicinchoninic acid method using BSA as the standard. The TGF-β1 levels of cell homogenate were assayed using a specific TGF-β1 enzyme-liked immunosorbent assay kit (Boster, Wuhan, China), and human TGF-β1 was used to construct a standard curve. The amount of TGF-β1 protein in the cell was normalized to the total amount of cellular protein. Absorbance values were read at 450 nm by an ELISA enzyme-labeled device.Statistical analysisStatistical analysis of data was performed by one-way ANOVA and a Student–Newman–Keuls test to determine statistically significant differences (p<0.05) between uninfected and HSV-1 infected cells.ResultsMorphological changes of HSV-1 infected human corneal epithelial cellsCell morphological changes were observed under phase-contrast microscopy. Normal HCE cells exhibited a typical cobblestone appearance (Figure 1A). Following HSV-1 infection and up to 8 h p.i., the cell morphology of infected groups was similar to the uninfected group. Compared with control cells, a cytopathic effect (CPE) in HCE cells could be observed at 8 h and 12 h p.i. (Figure 1B,C). The space between infected cells increased, and the cobblestone appearance disappeared. At 24 h p.i., most of the infected cells exhibited a CPE (dead cells were observed floating in the medium), and many giant multinucleated cells could be seen (Figure 1D).Figure 1Morphological changes of human corneal epithelial cells infected with HSV-1. A: Normal human corneal epithelial cells exhibited a cobblestone appearance. B: The cytopathic effect could be seen at 8 h p.i. The space between infected cells increased. After cells were infected with HSV-1 for 12 h (C) and 24 h (D), the cobblestone appearance disappeared and many giant multinucleated cells could be seen. Magnification: 200X.Expression of TGF-β isoforms in HSV-1 infected human corneal epithelial cells in vitroFirst, the mRNA level of TGF-β isoforms (i.e., TGF-β1, TGF-β2, and TGF-β3) in HCE cells infected with HSV-1 was estimated using RT–PCR (Figure 2). The mRNA level of TGF-β1 decreased significantly by 40.3%, 57.3%, and 70.4% at 8 h, 12 h, and 24 h p.i., respectively, when compared with uninfected cells (p<0.01). However, mRNA profiles of TGF-β2 and TGF-β3 in infected cells at 8 h, 12 h, and 24 h p.i were similar to that of uninfected cells (p>0.05).Figure 2Reverse transcription polymerase chain reaction analysis of the TGF-β isoforms in human corneal epithelial cells infected with HSV-1. A: Products of RT-PCR that were run on 2% agarose gel electrophoresis. The intensities of TGF-β1 bands decreased significantly at 8 h, 12 h, and 24 h p.i., while that of TGF-β2 and TGF-β3 bands unchanged. GAPDH was used as an internal control. B: The level of mRNA was expressed as the ratio of integrated optical density (IOD) of specific PCR products over GAPDH IOD. Each data was the mean value of three independent experiments. Single asterisks indicate significant differences (p<0.05).To further verify the results of PCR, indirect immunofluorescence was used to observe the changes of TGF-β1and TGF-β2 protein expression in HCE cells infected with HSV-1 (Figure 3). The intensity of immunostaining for TGF-β1 decreased at 12 h and 24 h p.i. compared with the control (Figure 3A). The decrease of TGF-β1 protein in HSV-1 infected HCE cells was also found by ELISA measurement (Figure 3C). Significant decreases in the levels of TGF-β1 protein were observed using two immunomethods. However, compared with normal cells, TGF-β2 protein remained present in infected cells at both 12 h and 24 h p.i. when we examined the cells by immunocytochemical staining (Figure 3B).Figure 3Protein expression of TGF-β1 and TGF-β2 in human corneal  epithelial cells infected with HSV-1. In A and B, indirect  immunofluorescence analysis was used to find the protein expression in  cells. FITC marked the secondary antibody (green; left), and PI dyed the nucleus (red; middle). Merged images were showed at the right of A and B. Scale bar: 10 μm. C: The expression of TGF-β1 by ELISA in HCE cells infected with HSV-1 was measured at 0 h, 12 h, and 24 h p.i. Significant decreases of the TGF-β1 protein in cell lysates were seen in both 12 h and 24 h post-infected cells (p<0.05). Each data was the mean value of four independent assays.Expression of SMAD2 and SMAD3 in HSV-1 infected HCE cellsThe expression of SMAD2 and SMAD3 in HCE cells infected with HSV-1 was detected by RT–PCR (Figure 4). This study found a clear reduction in mRNA level coding for SMAD3 in HSV-1 infected cells. Compared with normal cells, SMAD3 mRNA levels decreased significantly by 37.5% (12 h p.i.) and 53.1% (24 h p.i.; p<0.01) in infected cells. However, the mRNA levels of SMAD2 remained unchanged during the course of infection (p>0.05).Figure 4Reverse transcription polymerase chain reaction analysis of SMAD2 and SMAD3 in human corneal epithelial cells infected with HSV-1. A: Agarose gel pattern of RT–PCR products. The band intensities of SMAD3, not SMAD2, decreased during the period of post-infection. GAPDH was used as an internal control. B: The level of mRNA was expressed as the ratio of IOD of specific PCR products over the GAPDH gene IOD. The mean values of three independent experiments are shown. Single asterisks indicate significant differences (p<0.05).To examine whether the down-regulation of SMAD3 mRNA also results in a reduction in protein level, SMAD3 and phospho-SMAD3 protein expressions during HSV-1 infection were analyzed by immunocytochemistry. Compared with normal cells, protein expression of SMAD3 and phospho-SMAD3 in infected cells was weaker at 12 h and 24 h p.i. (Figure 5).Figure 5Colocalization of SMAD3 and phospho-SMAD3 protein in human corneal epithelial cells. FITC marked the secondary antibody (green; left), and PI dyed the nucleus (red; middle). Merged images were showed at the right of A and B. Both SMAD3 (A) and phospho-SMAD3 (B) were more weakly expressed at 12 h and 24 h p.i. compared to the uninfected cells. Scale bar: 10 μm.DiscussionThe cornea contains three principal cell types, epithelial cells, keratocytes, and endothelial cells. Previous studies have shown that corneal epithelial cells are capable of supporting efficient HSV-1 replication [26,27]. Balliet et al. [28] reported that a recombinant HSV-1 virus, KOS-CMVGFP, expressing enhanced green fluorescent protein (EGFP) could infect mice as efficiently as a wild-type virus. They found that fluorescence was observed in eyes as distinct small foci on the cornea at day 1 p.i., and that the fluorescence spread throughout the eye between days 1 and 3 p.i. Finally, the foci grew larger and coalesced, resulting in large, dendritic corneal lesions. Consistent with the studies described above, our work also demonstrated that the HCE cell is highly permissive to HSV-1 infection in vitro. When HCE cells were infected with HSV-1 at a MOI of 5, a cytopathic effect was observed at 8 h p.i. HSV-1 infection caused an increase in the number of dead cells, which may be the reason for the dendritic keratitis observed in vivo. Furthermore, we also observed expression of two viral genes (DNA polymerase and VP16) in infected cells by RT–PCR, which implies that HSV-1 caused a productive infection of HCE cells. Therefore, HCE cells are susceptible to HSV-1 infection, and it can provide a useful in vitro model for research of HSV-1 infection in the cornea.TGF-β isoforms and SMAD family members have been identified in mammalian cells. In the eye, TGF-β isoforms are expressed in different ocular tissues [14,15,29]. In the cornea, SMAD2 and SMAD4 were expressed and translocated into the nuclei, and SMAD7 was overexpressed during corneal epithelial wound repair [16,30]. In the cultured retinal pigment epithelial cell line (D407), TGF-β can stimulate the translocation of SMAD2 (but not SMAD1) from the cytoplasm into the nuclei [31]. Therefore, TGF-β isoforms and SMADs may play important roles in the pathogenesis of ocular diseases. However, there is limited research on the effect of TGF-β isoforms and SMADs in cells infected by HSV-1. Accordingly, the objective of the present study was to investigate whether the expression of TGF-β isoforms and SMADs in HCE cells is affected by HSV-1 infection in vitro.The effect of viral infection on TGF-β expression has been described for several viruses including HIV, CMV, and HSV-1 in other tissues [19,32,33]. In CMV infection, TGF-β1 was detected in increasing amounts in infected human foreskin fibroblast and astrocyte supernatants, and TGF-β1 transcription was significantly increased when compared to that of uninfected cells [22,33]. In vitro HSV-1 infection of human mononuclear cells resulted in a significant time-dependent increase in the release of TGF-β1 protein into supernatants [19]. These studies showed that virus infection could induce the overexpression of TGF-β1 with respect to protein expression and mRNA levels. However, in a study on mouse cornea infected with HSV-1, Hu et al. [4] showed that levels of TGF-β mRNA decreased in inflamed corneas. Our study demonstrated that the expression of TGF-β1 at both the mRNA and protein level was down-regulated in HCE cells infected by HSV-1 at 8 h p.i. and beyond. However, during the course of HSV-1 infection, the transcription of TGF-β2 and TGF-β3 remained unchanged compared to uninfected cells. These results suggested that TGF-β expression in response to HSV-1 infection is specific to cell type.The current study also showed that HSV-1 infection caused a decline in the transcription of SMAD3 in HCE cells but had no effect on the expression of SMAD2. Similarly, by confocal laser scanning microscopy, HSV-1 infected HCE cells displayed weak immunostaining for SMAD3 and phospho-SMAD3. Although measuring protein levels with a quantitative method such as western blot would provide more convincing evidence of protein expression change, the immunostaining result was consistent with the data of RT–PCR analysis for SMAD3. These results suggested that SMAD3 decreased in both mRNA and protein levels in HSV-1 infected HCE cells.It has been demonstrated that in virus infections, TGF-β could be regulated by the SMAD subfamily. In HPV infected cells, viral E7 oncoprotein blocks through its constitutive interactions with SMAD2, SMAD3, and SMAD4, both SMAD transcriptional activity and the ability of TGF-β to inhibit DNA synthesis [10]. E6 oncoprotein of HPV-5 inhibits SMAD3 transactivation by interacting with SMAD3, destabilizing the SMAD3/SMAD4 complex, and inducing the degradation of both proteins [34]. Virus proteins also interfere with TGF-β signaling via SMAD proteins as observed in HTLV-1 infected ATL cells [8] and in Kaposi's sarcoma-associated herpes virus infection [11]. These results show that suppression of SMAD-mediated TGF-β signaling in virus infected cells might contribute to the carcinogenesis. The present study focuses on HSV-1 infected corneal epithelial cells, which characterizes recurrent inflammation of the cornea in vivo. The fundamental physiologic roles of SMAD3 are involved in the processes of tissue repair and fibrosis [35]. Decreased SMAD3 expression could reduce formation and nuclear import of transcriptionally active SMAD heterocomplexes and decrease transcription of TGF-β1 regulated target genes, which result in reduced inflammatory cell infiltrates, reduced auto-induction of TGF-β, and reduced elaboration of collagen [36]. This may be the cause of the observed decreases of TGF-β1 and SMAD3 in HSV-1 infected HCE cells in this study, which occurred as an in vivo inflammatory process.The interplay between HSV-1 and its host involves numerous factors, and the virus employs several mechanisms to combat many antiviral responses enacted by the infected cell [37]. Expression of TGF-β1 and SMAD3 in HSV-1 infected HCE cells decreased in this study, which suggested that they may be involved in corneal diseases that are associated with HSV-1 infection. The specific function of TGF-β1 and SMAD3 in HSV-1 corneal infection requires further investigation.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2530867",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2530867\nAUTHORS: Agathe Bourgogne, Danielle A Garsin, Xiang Qin, Kavindra V Singh, Jouko Sillanpaa, Shailaja Yerrapragada, Yan Ding, Shannon Dugan-Rocha, Christian Buhay, Hua Shen, Guan Chen, Gabrielle Williams, Donna Muzny, Arash Maadani, Kristina A Fox, Jason Gioia, Lei Chen, Yue Shang, Cesar A Arias, Sreedhar R Nallapareddy, Meng Zhao, Vittal P Prakash, Shahreen Chowdhury, Huaiyang Jiang, Richard A Gibbs, Barbara E Murray, Sarah K Highlander, George M Weinstock\n\nABSTRACT:\nA comparison of two strains of the hospital pathogen Enterococcus faecalis suggests that mediators of virulence differ between strains and that virulence does not depend on mobile gene elements\n\nBODY:\nBackgroundEnterococci have emerged over the past few decades as the second to third most common cause of nosocomial infections, including urinary tract and soft tissue infections, bacteremia, and endocarditis [1-3]. They are well equipped to thrive in environments with heavy antibiotic usage due to both their intrinsic resistance to antibiotics and their talent for swapping genetic information, which allows them to gain and share resistance determinants. Entecococcal infections are predominantly caused by E. faecalis and E. faecium. However, many, if not most, strains of these species are harmless commensals, with some enterococci being marketed in Europe to alleviate symptoms of irritable bowel syndrome and recurrent chronic sinusitis or bronchitis (Cylactin® and Fargo688® (E. faecium) and Symbioflor 1 (E. faecalis)). To differentiate the two faces of this organism, genome-wide comparisons are necessary. Although hundreds of microbial genomes have been sequenced, only two E. faecalis genomes have been reported (V583 as a clinical isolate [4] and Symbioflor 1 as a commensal isolate [5]), but only the V583 genome has been made publicly available. In this strain, more than one-quarter of the genome is mobile DNA, more than any other sequenced bacterial genome [4]. The occurrence of multiple antibiotic resistance determinants in V583 [6] makes it difficult to manipulate genetically. Moreover, the vancomycin resistance phenotype makes this strain more of a risk to handle in the laboratory. To avoid these issues, most laboratories use strain OG1 or its close derivatives. OG1 is a human isolate subsequently shown to cause dental caries in rats [7]. OG1RF is a rifampicin and fusidic acid resistant derivative of OG1 [8,9]. By pulsed-field gel electrophoresis, Murray et al. [10] estimated the size of the OG1RF genome as 2,825 kb and created a restriction map of the chromosome. Multilocus sequence typing (MLST) showed that OG1RF is clonally distinct from V583 (differs in six out of seven alleles of housekeeping genes) [11] and characterization of regions flanking transposon insertions in OG1RF suggested that approximately 10% of their sequences differed [12].OG1 and its derivatives have been successfully used over the past 20 years in various animal models, starting with the demonstration that it can cause caries in germ-free rats [7], and later to characterize factors important for E. faecalis virulence in a mouse model of peritonitis [13], a rabbit model of endophthalmitis [14], a rat model of endocarditis [15] and in a mouse urinary tract infection model [16]. OG1RF was also shown to be as virulent as V583 in the model host Caenorhabditis elegans [17]. In addition to its virulence, the main reasons for the extensive use of OG1RF as a laboratory strain are that it does not carry plasmids, is readily transformable by electroporation, and is not resistant to commonly used antibiotics, other than rifampicin and fusidic acid. These resistances were serially selected in OG1 to provide strain markers [9]. The lack of resistance to common antibiotics facilitates the selection of plasmids, transposons, and allelic replacement markers introduced into the strain.Numerous factors important for virulence have been characterized in OG1RF. A recently described example are the Ebp pili, whose subunits are encoded by the ebp operon [18] and whose genes are regulated by EbpR [19]. A non-piliated mutant produces less biofilm than the parent strain and is attenuated in a rat model of endocarditis [18] and in a murine urinary tract infection model [16]. Also present is Ace, a member of the MSCRAMM (microbial surface component recognizing adhesive matrix molecules) family. The ace gene, like the ebp locus, is ubiquitous in E. faecalis and it occurs in at least four different forms that vary in the number of repeats of the B domain [20]. Ace mediates conditional (that is, after growth at 46°C or in the presence of serum or collagen) adherence of E. faecalis to collagen type IV and to laminin [21] and, in unpublished data, influences the ability of OG1RF to cause experimental endocarditis (KV Singh and BE Murray, unpublished observation). Finally, the Fsr system, a major positive and negative transcriptional regulator in OG1RF [22], affects expression of several virulence factor genes, including gelE, which encodes gelatinase [23], and contributes to infection in various animal models [15,24].The distinct MLST profile and the wide range of phenotypic and genotypic analyses of OG1RF, including many molecular genetic studies and experiments in various animal models, suggested that genomic analysis of this strain would prove insightful and would be useful to future studies. Thus, we analyzed the sequence of E. faecalis OG1RF. This revealed approximately 232 kb encoding 227 open reading frames (ORFs) that are unique to this important strain compared to V583. The unique regions were then characterized further.Results and discussionGeneral genome featuresThe complete circular chromosome of OG1RF was found to be 2,739,625 bp with an average G+C content of 37.8%. The complete OG1RF sequence was obtained using three independent techniques (Solexa, the 454, and Sanger sequencing technique) with a higher than classic coverage (more than 100 times), diminishing the likelihood of sequencing-related frameshifts, base errors and/or misassembly. A comparison of our assembly of the closed OG1RF genome with the restriction map of OG1RF published by Murray et al. [10] showed only minor variations (primarily an overestimation of 30 kb for the Sfi I fragment E, 540 kb versus 509 kb predicted from the sequence; Figure 1).Figure 1Map of the OG1RF chromosome. The following features are displayed (from the inside out): restriction maps using SfiI, AscI, and NotI (black) from Murray et al. [10] overlaid with the digestion profile predicted from the sequence (red); G+C content in percentage in green; the total OG1RF-unique genes are shown in purple with those in (+) orientation labeled in blue, and those in (-) orientation labeled in red.We found 232 kb of OG1RF unique sequences distributed in 48 regions ranging from 101 bp to approximately 49 kb in length (Figure 1; Additional data file 1). Using the published DNA sequence of V583 as reference (NC_004668), OG1RF shares 2,474 ORFs as well as the 12 rRNA genes and 58 of 68 tRNA genes (Table 1). The 10 missing tRNA are localized in a region in V583 that has been replaced in OG1RF by a 49 kb region (see below). Surprisingly, the genomes align syntenically, as shown in Figure 2, despite the fact that 25% of the V583 genome is composed of mobile elements. Similarly, the presence of OG1RF-unique sequences has not affected the overall chromosomal arrangement. Some of the major insertions/deletions in the two genomes are shown in Figure 2, such as the absence of the pathogenicity island (PAI) in OG1RF and the presence of an approximately 49 kb fragment unique to OG1RF. However, most of the differences are small and cannot be visualized in this figure. Overall, we found 64 areas of divergence between the genomes that can be divided into 3 classes: an additional sequence present in OG1RF when compared with V583; a sequence replacement where a sequence in OG1RF differs from the sequence in V583; and the absence of a sequence from OG1RF when compared with V583.Table 1General features of OG1RF compared to V583V583OG1RFGeneral featuresSize (base pairs)3,218,0312,739,633G+C content (%)37.537.8rRNA genes1212tRNA genes6858Genes common to both strains2,474*Genes unique to OG1RFSimilar to known proteins114†Conserved hypotheticals50No database match63Total227Total number of ORFs3,1132,701‡*The assessment of the genes common to both strains is based on the homology at the DNA level with the ORFs described for V583 (source TIGR [70]). The BLASTN cutoff e-value was 1e-5. †This number includes the proteins with domain polymorphism (see text for details). ‡Estimated number of ORFs calculated by adding the OG1RF-unique ORFs to the number of ORFs shared with V583.Figure 2Dot plot of OG1RF versus V583 generated by BLASTN. The dot plot was generated by aligning the OR1RF genome against the V583 genome using BLASTN (e-value 1e-10). The alignment pairs were plotted according to their genome coordinates. The visible areas of divergences are labeled using 'Δ ' to indicate a sequence absent in OG1RF and '∇ ' to indicate a sequence unique to OG1RF (locus tag OG1_xxxx) when compared with V583 (locus tag EFxxxx). Phages 1, 3, 4, 5, 6, 7 of V583 (φ1 to 7; see [31]) and the PAI locations, all of which are missing from OG1RF, are also indicated.CRISPR lociThe CRISPR (comprised of regularly interspaced short palindromic repeats) loci encoded by some bacterial strains is a recently described system that protects cells from infection with bacteriophage [25-27]. The specificity of the phage resistance conferred by the CRISPR elements and CRISPR-associated genes (cas genes) is determined by spacer-phage sequence similarity. OG1RF carries two CRISPR elements: CRISPR1 (between the OG1RF homologue of EF0672 and EF0673) and CRISPR2 (between the OG1RF homologue of EF2062 and EF2063); CRISPR1 is linked to cas-like genes while CRISPR2 is not (Figure 3). Both OG1RF CRISPR elements are composed of 7 repeats of a 37 bp palindromic sequence with a 29 bp spacer. None of the 29 bp spacers (14 total) have homology to any sequences in GenBank. The CRISPR1-associated proteins belong to the Nmeni subtype [28]. Species bearing this CRISPR/cas subtype have so far been found exclusively in bacteria that are vertebrate pathogens or commensals. The Nmeni subtype is characterized by the presence of four specific cas genes and a single copy of the repeat that is upstream of the first gene in the locus. The four cas genes encode Cas_csn1 (possible endonuclease), Cas1 (novel nuclease), Cas2 (conserved hypothetical protein), and Cas_csn2 (conserved hypothetical protein). The repeat upstream of cas_csn1 appears to have degenerated since it shares only 23 bp with the 37 bp repeat cluster downstream of the last gene. A unique feature of the OG1RF CRISPR1 locus is the presence of a gene downstream of the element, which encodes a hypothetical 119 amino acid transmembrane protein.Figure 3The two CRISPR loci of OG1RF. (a) The CRISPR1 locus. The CRISPR1 element is represented with a hatched box while the CRISPR1 associated genes are represented in orange; the white arrows indicate ORFs present in both OG1RF and V583. The black diamonds represent the 37 bp repeat sequences, while the open boxes with a number indicate the 29 bp unique sequences. (b) The CRISPR2 locus containing only a CRISPR element. (c) CRISPR consensus and unique sequences. The underlined bases indicate mismatches at these locations. The sequences numbered 1 to 14 represent the unique sequences located in the CRISPR1 and CRISPR2 elements.The presence of the CRISPR loci among E. faecalis strains may be a powerful tool to avoid the load of prophage replication. To determine the distribution of the CRISPR1 locus in E. faecalis strains, 16 isolates of various MLST types were tested for the presence (PCR with primers specific for csn1 and cas1) or absence (PCR with primers overlapping the junction between EF0672 and EF0673) of the CRISPR1 locus (Table 2). Seven strains were cas positive, but negative for the junction and the remaining nine were positive only for the junction. This indicates that the location of the CRISPR1 locus appears to be conserved (between EF0672 and EF0673 when compared with the V583 genome). Interestingly, the two vancomycin resistant strains tested were both cas negative. It is appealing to postulate that the presence of the CRISPR locus in OG1RF may be the reason for the absence of prophage in this strain.Table 2Frequency of the CRISPR locus among E. faecalisNameOtherOriginSource/referenceMLSTErmR*VanR†cas‡EF0672-3§TX4002OG1RFHuman[8,9]1--+-TX2708V583Clinical isolate[6]6¶++-+TX2144E1840Clinical isolateRuiz-Garbajosa P.#40+-+-TX2135E1795Hospital surveyRuiz-Garbajosa P.44---+TX2137E1798Hospital surveyRuiz-Garbajosa P.16+-+-TX2141E1825Clinical isolateRuiz-Garbajosa P.25---+TX2140E1803Hospital surveyRuiz-Garbajosa P.38---+TX2138E1801Hospital surveyRuiz-Garbajosa P.48---+TX2146E1844Clinical isolateRuiz-Garbajosa P.61---+TX2139E1802Hospital surveyRuiz-Garbajosa P.35+-+-TX4240A0826PigJensen L.98+-+-TX4247E1876PigGaastra W.20+-+-TX4245E1872DogGaastra W.16+-+-TX4243E0252CalfMevius D.23++-+TX4255A0808Clinical isolateKawalec M.88---+TX4259A1006Clinical isolateKawalec M.135---+*Erythromycin resistance was tested at 5 μg/ml. †Vancomycin resistance was tested at 10 μg/ml. ‡Two sets of primers were used to detect the cas genes (cas1 and csn1). §This set of primers amplifies the junction between EF0672 and EF0673 where the CRISPR1 locus is inserted in OG1RF. ¶CC2. # Ruiz-Garbajosa P. (Spain), Jensen L. (Denmark), Gaastra W. and Mevius D. (Netherland), and Kawalec M. (Poland).A 14.8 kb region inserted in the 23.9 kb region containing fsrA and fsrBNakayama et al. [29] described a conserved 23.9 kb chromosomal deletion when comparing fsrA-lacking/fsrC+/gelE+ strains (by PCR) from various origins with V583; the deleted sequences start in the middle of EF1841, include the fsrAB genes and end in the middle of the fsrC gene (EF1820). Loss of the fsr regulatory components results in a gelatinase-negative phenotype under routine test conditions despite the fact that these strains still carry the gelE gene [23,29]. The absence/presence of the 23.9 kb region, from EF1820/fsrC to EF1841, did not appear to correlate with the clinical origin of the isolates [30]. In a more recent analysis of relationships between various E. faecalis strains, the 23.9 kb region was not detected in 86% of the strains of the clonal complex (CC)2, 58% of the CC9 strains, nor in any of the CC8 strains [31]. The Symbioflor 1 strain, used as a probiotic, is one representative of the 7.4% of E. faecalis isolates that are missing the gelE gene in addition to the 23.9 kb region [5,30]. Our analysis of this area in OG1RF revealed the presence of an additional 14.8 kb fragment inserted between the corresponding EF1826 and EF1827 of OG1RF (confirmed by PCR; results not shown). In OG1RF, this 14.8 kb region contains two loci, a WxL locus (described below) and a seven-gene locus that may encode a possible ABC transporter with similarity to one annotated in Pediococcus pentosaceus.Components of the cell surfaceIt has been shown in E. faecalis that at least one cell surface protein (Ace) is subject to domain variation [20] and it has been postulated that domain variation may help bacteria escape the immune system. We found more polymorphisms in two families of E. faecalis proteins present on the cell surface: the MSCRAMMs and the WxL domain surface proteins. The MSCRAMMs are composed of two large regions, namely, the non-repeat A region (which is usually the ligand binding region for extracellular matrix molecules such as collagen or fibrinogen) and the B region (which typically contains repeated sub-domains). The B region of Ace contains five repeats in OG1RF, while it contains only four in V583 [20]. We found two other MSCRAMM proteins that show polymorphisms in the number of their B repeats. OG1RF_0186 (corresponding to EF2505 of V583) has four repeats compared to seven in V583, and OG1RF_0165 (corresponding to EF2224 of V583) has eight repeats compared to five in V583. It has been proposed that the repeats are used as a stalk that projects the A region across the peptidoglycan and away from the cell surface [32]. A hypothesis that the number of repeats may be proportional to the depth of the peptidoglycan has been proposed [32]. However, OG1RF_0186 carries fewer repeats than EF2505 while Ace and OG1RF_0165 carry more repeats than their counterparts in V583, suggesting that our observation does not fit this hypothesis or that the peptidoglycan depth is not uniform. Apart from these three MSCRAMMs with B-repeat polymorphisms, we identified two unique MSCRAMM proteins in OG1RF: a homologue of EF0089 (OG1RF_0063, which shares 48% similarity) and a homologue of EF1896 (OG1RF_0039, which shares 75% similarity); both are located in the approximately 49 kb region unique to OG1RF described below (Figure 1; Additional data file 1).Another family of E. faecalis surface proteins includes the newly described WxL domain surface proteins. Siezen et al. [33] reported a novel gene cluster encoding exclusively cell-surface proteins that is conserved in a subgroup of Gram-positive bacteria. Each gene cluster has at least one member of three gene families: a gene encoding a small LPxTG protein (approximately 120 amino acids); a gene encoding a member of the DUF916 transmembrane protein family; and a gene encoding a WxL domain surface protein. In addition, members of these gene families were found as singletons or associated with genes encoding other proteins (Additional data file 2). Recently, it was shown that the WxL domain attaches to the peptidoglycan on the cell surface [34] and one member of this WxL domain family, the homologue of EF2686 in OG1RF (a probable internalin protein), was shown to be important for virulence in a mouse peritonitis model and is required for dissemination to the spleen and liver [35]. OG1RF shares five complete WxL loci with V583 (EF0750-7, EF2682-6, EF2970-68, EF3181-8, and EF3248-53). OG1RF does not contain homologues of EF2248-54 (carrying instead the iol operon), though it has a novel WxL locus within the 14.8 kb unique region upstream of the fsr locus (Additional data file 2). In addition to the variation in the number of WxL loci, we also observed polymorphisms in six of the WxL domain surface proteins. For example, OG1RF_0213 shares 88% similarity with EF3188, while OG1RF_0224, OG1RF_0225, and OG1RF_0227 share 64-68% similarity with their V583 counterparts, EF3248, EF3250, and EF3252, respectively. Also, in place of EF3153, EF3154, and EF3155 (which share 70% similarity among themselves), were found non-distantly related homologues, OG1RF_0209 and OG1RF_0210, which share 60-80% similarity with EF3153, EF3154, and EF3155. It is interesting to note that while several of these WxL loci, including the EF0750 and EF3184 loci, were present by hybridization in all the strains (clinical or food isolates) tested by Lepage et al. [36], other loci, including the EF3153 and EF3248 loci, were not detected in the majority of these strains. In addition, it appears that the EF3248 locus diverges in the Symbioflor 1 strain. When compared to V583, the sequence identity in this area between the two strains appears to be as low as 75% (depicted in Figure 2 from reference [5]).However, because the Symbioflor 1 genome sequence is not currently available, it was not possible to compare their respective sequences in more detail. Since these proteins are located at the surface of the cell, the low level of homology shared between them may be the result of antigenic variation. More analyses are required for a better understanding of the number, frequency and function of these WxL domain proteins and their possible relationship with the diversity of E. faecalis.Finally, as previously found using PCR, the cpsCDEFGHIJK operon capsule polysaccharide genes [37] were confirmed here as missing, although OG1RF carries the cpsA and cpsB genes, which were proposed to be essential for E. faecalis since all strains tested by Hufnagel et al. [37] carry these two genes. In OG1RF, the region that would encode the cps operon is only 59 bp in length and has no homology with V583. Thus, while V583 and OG1RF share much similarity between their surface components, there are unique differences that could potentially be important in affecting the behavior of the strains and might be useful for strain typing.Two-component regulatory systemsOG1RF lacks four two-component systems found in V583. These are histidine kinase-response regulator (HK-RR)08, HK-RR12 located in the PAI, HK-RR16 and the vanB regulatory system HK-RR11 [38]. However, an OG1RF-unique two-component system with high homology with the vanG locus was found at the location corresponding to the region between EF2860 and EF2861 in V583 (Table 3). OG1RF_0193 shares 82% similarity with VanRG and 81% similarity with VanRG2. Similarly, OG1RF_0192 shares 68% similarity with VanSG and VanSG2. A gene (OG1RF_0191) encoding an M15 family muramoyl pentapeptide carboxypeptidase is located downstream of these two-component regulatory genes (Figure 4a). The predicted carboxypeptidase (OG1RF_0191) shares 69% similarity over 179 amino acids with EF2297, a membrane-associated D, D-carboxypeptidase encoded by the vanB operon in V583. However, OG1RF_0191 lacks an identifiable transmembrane domain that is important to the VanY function and it is likely, therefore, that this protein may be a soluble D, D-carboxypeptidase/transpeptidase as seen in Streptomyces [39] and Actinomadura [40], and thus may not be involved in peptidoglycan metabolism. Consequently, it seems unlikely that this operon is a remnant of a vancomycin resistance operon in OG1RF, but rather part of a still unknown regulatory pathway.Table 3OG1RF-unique regulatorsOG1RFDescriptionBest hitSize*CommentsOG1RF_0070Transcriptional regulator116512576102-OG1RF_0073LytR family response regulator81428169151-OG1RF_0120BglG family transcriptional antiterminator47095712494Probable regulator of the downstream PTS systemOG1RF_0138Transcriptional regulator116493423219Probable transcriptional regulator of the downstream ABC superfamily transporterOG1RF_0143GntR family transcriptional regulator82745913236Probable regulator of the downstream PTS systemOG1RF_0175DNA binding protein15890504293Probable regulator of the iol operonOG1RF_0192Sensor histidine kinase VanSG119635646371Best homology with VanG andOG1RF_0193Response regulator VanRG119635645235VanG2 two-component systems.OG1RF_0192 and OG1RF_0193 appear cotranscribed with a gene encoding a M15 family muramoylpentapeptide carboxypeptidaseOG1RF_0198Response regulator47567135240Best homology with AgrA from Bacillus cereus G9241. However, no presence of AgrB or AgrD homologues in the vicinity. Also similar to ComE of S. pneumoniae (52% similarity)OG1RF_0199Sensor histidine kinase47567134443Best homology with AgrC from Bacillus cereus G9241. Also similar to ComD of Streptococcus pneumoniae (48% similarity)OG1RF_0220Probable endoribonuclease MazF69244828114Toxin-antitoxin described in E.OG1EF_0221Probable antitoxin MazE6924482977coli and recently on an E. faecium plasmid*Amino acidsFigure 4Two-component systems unique to OG1RF. (a) Two-component system with homology to the VanG system. (b) Two-component system with homology to the comCD genes of S. pneumoniae. The two-component system (OG1RF_0198 and OG1RF_0199) is indicated in light blue; the two ORFs encoding potential transporter proteins (OG1RF_0200 and OG1RF_0201) are represented in pink. In green are indicated two small ORFs encoding polypeptides of less than 51 amino acids. The white arrows indicate ORFs also present in V583.The iol operonOG1RF carries an iol operon while V583 does not. This operon encodes the factors necessary for the degradation of myo-inositol into glyceraldehyde-3P. Many soil and plant micro-organisms, including Bacillus subtilis [41] (first iol operon identified), Klebsiella spp. [42], and cryptococci [43], have been reported to use myo-inositol as a sole carbon source. Myo-inositol, one of the nine isomers of the inositol group, belongs to the cyclitol group and is abundant in nature, particularly in the soil. The OG1RF iol operon appears to be closely related to ones described in Clostridium perfringens [44] and Lactobacillus casei [45]. In L. casei, the myo-inositol operon consists of ten genes with an upstream divergent regulator gene, iolR. In OG1RF, the operon appears to include ten genes, beginning with a probable transcriptional regulator (helix-turn-helix domain protein). Also, the OG1RF operon carries two copies of an iolG-like gene, which encodes inositol 2-dehydrogenase, the first enzyme of the myo-inositol degradation pathway (Figure 5). However, the order of the genes is not the same between E. faecalis and L. casei. In addition, iolH,iolJ and iolK, present in L. casei, are not present in OG1RF, nor are iolH and iolK present in the C. perfringens iol operon.Figure 5The iol operon. The iol genes are labeled based on the homology/conserved motif of their encoded proteins with known enzymes necessary for myo-inositol degradation. For all strains, the described or probable regulator is represented in blue. E. faecalis OG1RF: the iol operon is represented in yellow, OG1RF_0166 (green arrow) located downstream of the iol operon encodes a probable PTS IIC component, while the white arrows indicate ORFs also present in V583. For B. subtilis 168, C. perfringens strain 13, and L. casei BL23, the iol genes are represented in green, orange and purple, respectively. C. perfringens iol mRNA transcript includes five other genes encoding proteins whose functions do not appear to be related to myo-inositol degradation; these genes are represented in gray.Yebra et al. reported that L. casei was the sole member of the Lactobacillales to carry a functional iol operon [45]. To survey E. faecalis, also a member of this order, for the presence of the iol operon, 48 isolates with different MLST and/or from various origins (including OG1RF and V583) were tested for myo-inositol fermentation; 23 of 48 isolates were positive. In addition, PCR verified the presence of iolE and iolR in these strains and in one negative for myo-inositol fermentation, indicating that the iol operon is not unique to OG1RF. To verify that the iol genes are responsible for the fermentation of myo-inositol in OG1RF, transposon insertion mutants [9] in the iolB and iolG2 genes of OG1RF were tested. Both mutants failed to ferment myo-inositol (data not shown), demonstrating that these genes are essential for myo-inositol fermentation. To investigate whether the iol operon was 'inserted into' or 'removed from' a putative ancestral strain, the sequences surrounding the iol genes were examined. In OG1RF, the iol operon is located between the equivalent of EF2239 and EF2352 when compared with V583. In V583, this region encodes probable prophage proteins and carries the vanB transposon, which confers vancomycin resistance. Since we did not identify any remnants of the iol operon in V583, it would appear that at least two independent events at the same location differentiate OG1RF and V583, suggesting that it is a hot region for rearrangement. This region between EF2239 and EF2352 (111 Kb) is also missing in the Symbioflor 1 strain (referred to as gap 2) [5]. The possible junction and presence of unique sequence in this region, if investigated, was not mentioned in the publication. Nonetheless, preliminary analysis of other strains' genotypes in this area seemed to confirm the hypothesis of a hot region for rearrangement (data not shown).A homologue of Tn916 in OG1RFAn analysis of the G+C content of OG1RF unique regions revealed several loci with a lower G+C content than the 37.8% average content of OG1RF. One of these is an approximately 49 kb fragment with a G+C content of 32.1% located between an rRNA operon and the homologue in OG1RF of EF1053, replacing 10 tRNA genes present in V583 (Figure 1). This fragment appears to be a patchwork composed of hypothetical genes, homologues of Tn916-associated genes and homologues of genes from other Gram-positive organisms, including Listeria, E. faecium, staphylococci, or lactococci (Additional data file 1). It is interesting to note that this region contains: a putative adhesin protein gene (OG1RF_0039) at one end of the fragment; homologues of 14 Tn916-associated genes (Tn916_2 to Tn916_12, Tn916_18 and Tn916_19, with an average of 70% similarity); and a gene encoding a putative integrase (OG1RF_0088) at the other end - these three features are also present in Tn5386 in E. faecium D344R [46]. However, the approximately 49 kb fragment lacks an excisase gene and the probable lantibiotic ABC transporter genes present in Tn5386.An uninterrupted competence operon in OG1RFOG1RF contains what appears to be an intact competence operon while that of V583 appears to be non-functional. This operon in OG1RF is similar to a nine-gene operon described in Streptococcus mutans [47], as shown in Figure 6. For example, the homologue in OG1RF of EF2046 shares 61% similarity with ComYA and the OG1RF homologue of EF2045 is 55% similar to ComYB. In S. mutans, only the first seven genes of the operon are essential for competence [47]. In V583, the fourth gene of this operon (corresponding to OG1RF_0148) is interrupted by phage 4 (EF1896-EF2043); in addition, EF1984 contains a premature stop codon not found in the corresponding gene in OG1RF (OG1RF_0228).Figure 6The OG1RF competence operon and its similarity with the competence operon of S. mutans. The ORFs essential for natural competence in S. mutans are shown in green as well as their homologues in OG1RF and V583. The ORF corresponding to the homologue of ComYD was not described in V583 [4], due to the presence of a probable prophage (EF1986-EF2043). The premature stop codon in EF1984 in V583 is indicated with an asterisk. ackA/EF1983 is represented in orange. The proteins encoded by the ORFs represented in white do not share any features of the known competence proteins or homology between S. mutans and E. faecalis; in S. mutans, ackA and ytxK are co-transcribed with the comY genes [47].Natural competence has not been reported for E. faecalis. To assess the functionality of this operon in OG1RF, we evaluated the competence of cells in different phases of growth (early log growth to stationary phase) using pAM401 [48] and pMSP3535VA [49]. We were not able to show natural competence under the conditions tested. We have also noted that V583 is less transformable by electroporation than OG1RF. To investigate the possibility that directly or indirectly the com operon might be responsible for this phenotype, we also evaluated transformability by electroporation. When compared with OG1RF, transposon mutants [12] in the OG1RF equivalent of EF2045 (encoding the comGB homologue) and in the OG1RF equivalent of EF1986 (encoding the comGF homologue) showed similar levels of transformability by electroporation (data not shown), implying that the difference in electroporation efficiency observed between OG1RF and V583 is not related to this locus.In Streptococcus pneumoniae [50], the competence operon is tightly regulated by a quorum sensing two-component system (ComDE) and a competence-stimulating peptide (CSP; encoded by comC). We did not find any homologues of CSP in OG1RF. Two homologous ComDE sensor histidine kinase/response regulators were found in OG1RF, one of which is FsrC/FsrA. Based on our previous microarray data, the Fsr system does not regulate the comY operon, at least under our previously used conditions (mid-log phase growth to early stationary phase in brain heart infusion (BHI)) [22]. The other ComDE homology is that with a two-component system unique to OG1RF (OG1RF_0199 and OG1RF_0198, respectively) that lies on a 4,706 bp unique fragment that maps between EF3114 and EF3115 in V583. This fragment also carries two genes (OG1RF_0200 and OG1RF_0201) encoding homologues of the YhaQ and YhaP sodium efflux ATP-binding cassette efflux/transporter proteins (Figure 4b). Although they are potential elements of a secretion apparatus, these two proteins do not share any homology at the protein level with the competence secretion apparatus ComAB of S. pneumoniae [51] nor CslAB from S. mutans [52]. Searching for a possible CSP in the vicinity of these genes, we identified a small ORF encoding 50 amino acids between yhaP and OG1RF199 and another encoding 20 amino acids downstream of OG1RF198. More analysis will be required to determine if there are conditions in which the OG1RF com operon is expressed and to determine whether or not this two-component system is involved in competence.Limited presence of mobile elementsBy probing a microarray of the V583 genome and plasmids with OG1RF genomic DNA, we previously estimated that only 75% of V583 ORFs were also present in OG1RF [22]. Later, Aakra et al. [53] compared nine strains, including OG1RF to V583, using comparative genomic hybridization. In these results, OG1RF appears to carry a few genes included in the PAI, and a few prophage genes. Using the complete genome sequence, we have now found that OG1RF lacks 639 genes and the three pTET plasmids described in V583. All but 45 of the missing genes are associated with putative mobile elements, such as the entire PAI, the recently described phages 1, 3, 4, 5, 6, and 7 [31], and the approximately 111 kb area between genes EF2240 and EF2351 (including the vanB transposon) present in V583. The absence of these elements appears also to be a characteristic of the commensal strain Symbioflor 1, although because the genome was not completely finished, the possibility remains that some of these regions were not sequenced. In conclusion, other than the approximately 49 kb fragment containing a Tn916 homologue, it appears that OG1RF has only one additional possible mobile element derivative, namely the phage 2 proposed to be part of the core genome [31].Fusidic acid and rifampicin resistanceOG1RF was sequentially selected from OG1 for resistance to fusidic acid and rifampicin [9]. The mutation leading to rifampicin resistance was identified in the rpoB gene by Ozawa et al. [54] and is caused by the A1467G mutation, which results in substitution of arginine for histidine at amino acid 489. The mutation also affected the clumping phenotype of traA mutants and this effect appears to be specific for the pAM373 system [54]. All of the other 22 differences in rpoB between OG1RF and V583 are synonymous. Fusidic acid resistance is associated with mutation(s) in the fusA gene, which encodes elongation factor G. We compared fusA from OG1RF with that in V583 and identified two differences (C1368A and T1992C). The mutation T1992C is synonymous, while the mutation C1368A leads to the presence of glutamine (histidine in V583) at position 404 in OG1RF. Mutations in this region have been associated with fusidic acid resistance in Staphylococcus aureus [55,56], and thus the C1368A mutation is likely the cause of the fusidic acid resistance phenotype in OG1RF.Virulence and biofilm comparisons of OG1RF with V583When compared in the mouse peritonitis model, the LD50 values of V583 in different determinations were lower (4.8 × 107 to 1.1 × 108 colony forming units (CFU)/ml) than the LD50 values of OG1RF (1.2 × 108 to 4.8 × 108 CFU/ml). However, at comparable inoculum, OG1RF (4 × 108 CFU/ml) showed more rapid mortality versus V583 (5 × 108 CFU/ml) in the first 48 hours (P = 0.0034; Additional data file 3). In a urinary tract infection model administering mixed equal inocula of V583 and OG1RF, OG1RF significantly outnumbered V583 in kidney with geometric means of 1.3 × 104 CFU/gm for OG1RF versus 1.9 × 102 CFU/gm for V583 (P = 0.0005); in urinary bladder homogenates, the geometric mean CFU/gm was 1.7 × 103 for OG1RF versus 6.6 × 101 for V583 (P = 0.003; Figure 7a). Similarly, in mono-infection, the geometric mean CFU/gm of OG1RF in kidneys was 9.4 × 103 versus 4 × 101 for V583 (P = 0.0035; Figure 7b). We also found that OG1RF produced 20% more biofilm (P < 0.01) than V583 at 24 hours (results not shown). These results, together with the previous results in C. elegans [17], demonstrate that OG1RF, although lacking what was thought to be important for virulence (PAI, plasmids, prophages), is as pathogenic as V583 in at least three assays.Figure 7Comparison of OG1RF and V583 in a mouse urinary tract infection model. (a) Mixed infection by wild-type E. faecalis strains OG1RF and V583 in the kidneys and urinary bladders of mice (n = 21; competition assay). Data are expressed as the log10(CFU)/gm for OG1RF or V583; the log10(CFU)/gm for both kidneys were combined and averaged from two independent experiments. Black solid diamonds and triangles represent E. faecalis strains OG1RF and V583, respectively, for kidney homogenates, and empty diamonds and triangles represent OG1RF and V583, respectively, for urinary bladder homogenates. Horizontal bars represent geometric means. Log10(CFU) were compared for statistical significance by the paired t-test. The minimum detection limit in these experiments was 101 and 102 CFU/gm of kidney and urinary bladder homogenates, respectively. (b) Mono-infection using E. faecalis strains OG1RF or V583 in the kidneys of mice (103 CFU per mice, n = 9). Data are expressed as log10(CFU)/gm for OG1RF recovered from kidney homogenates 48 h after infection; the log10(CFU)/gm for a kidney pair were combined and averaged. Black and white triangles represent OG1RF and V583, respectively. Horizontal bars represent geometric means. The CFU of V583 recovered from kidneys was significantly reduced compared to the CFU of OG1RF, as determined by the unpaired t-test.ConclusionE. faecalis OG1RF carries a number of unique loci compared to V583. Those of particular interest include new surface proteins (MSCRAMMs and WxL domain proteins), an operon encoding myo-inositol utilization, an intact competence operon, and two CRISPR elements. The CRISPR elements may be of particular significance when one considers that most of what is missing from OG1RF compared to V583 consists of mobile genetic elements (MGEs), including 6 phages or remnants thereof. The presence of the CRISPR elements in OG1RF provides a tantalizing, but as yet unproven, explanation for the discordance in the number of mobile elements between these two strains.The acquisition of MGE is believed to be an important mechanism by which the species E. faecalis had been able to generate genetic diversity and, thereby, highly variable phenotypes [4]. It has been proposed that the ability of E. faecalis to cause healthcare related infections is associated with these MGEs [4,5]. This hypothesis was supported by several studies that have highlighted the importance of virulence determinants carried by these mobile elements, such as cytolysin [57] by the PAI. However, more recent results from Aakra et al. [53] and Lepage et al. [36] demonstrate that these factors may be present in harmless strains while absent in clinical isolates, indicating that E. faecalis virulence is not dependent on any single virulence factor. Indeed, few studies have compared the virulence pattern of strains from various origins. The increased ability of OG1RF to infect kidneys and to produce biofilm, despite the absence of MGEs and their associated virulence factors, was surprising. Different possibilities can be proposed relating to the factors important for these differences in enterococcal infections. One of these is that virulence in the assays used may be linked to the shared core genome of these two strains, with the differences arising from the unique genes. On the other hand, virulence could be associated primarily with the genes unique to each strain, but with each set being able to complement the absence of the other. It seems most likely that virulence, and/or some combination of virulence and fitness, is caused by the expression of a mixture of both the core and unique genes. It is also important to remember that E. faecalis is a well adapted commensal, carrying the genes necessary to survive and to colonize the gut, and that a subset, particularly MLST CC2 and CC9 [58], predominates among hospital acquired infections. It may be that these clonal complexes are not more virulent per se, as defined in the assays described here, but rather are better able to survive and/or colonize hospitalized patients, taking advantage of factors that predispose to nosocomial infections such as urinary or venous catheters, or mucositis, among others.Sequencing of more E. faecalis strains may facilitate our understanding of the path from commensalism to pathogenicity, a crucial prerequisite for designing therapeutic interventions directed to control an organism that is already resistant to a large spectrum of antibiotics.Materials and methodsStrainsE. faecalis OG1 is a strain of human origin (formerly designated 2SaR [7]) and was subsequently selected on rifampicin and fusidic acid [8,9] to generate OG1RF (deposited at the American Type Culture Collection (ATCC) under ATCC accession number 47077). V583 is a vancomycin resistant E. faecalis strain [6], recovered from a blood culture of a patient hospitalized at the Barnes Hospital, St Louis, MO, USA in February 1987 (ATCC accession number 700802, NCBI complete genome accession number NC_004668). Bacteria were grown routinely at 37°C in BHI broth (Difco Laboratories, Detroit, MI, USA) or BHI agar unless otherwise indicated. Comparisons of OG1RF and V583 grown in broth (BHI, tryptic soy broth with glucose (TSBG), or BHI with 40% serum) did not reveal any obvious differences.DNA sequencing and genome assemblyGenomic DNA was purified from cesium chloride (CsCl) gradients of whole cell lysates [10]. DNA sequencing was performed by a combined approach using 454 Life Sciences pyrosequencing strategies [59] and the Solexa approach [60]. Read-pair information was used to create higher order scaffolds. Sanger sequencing was used for OG1RF whole gun sequencing and finishing. The coverage was 28× by the 454, 104× by Solexa, and 4.5× by Sanger sequencing technique. The assembly was done using Atlas [61].Gene identification and annotationGene prediction and manual annotation were performed as previously described [62]. Glimmer [63] and GeneMark [64] were used independently to predict ORFs. Visualization of gene predictions was performed using the Genboree system [65] and the CONAN database [66]. OG1RF-unique ORFs were analyzed with BLASTN and BLASTX. Protein sequences were analyzed by BLASTP versus the nr database at NCBI [67]. When appropriate, other predictive tools were used as described previously [62]. This whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the project accession ABPI00000000. The version described in this paper is the first version, ABPI01000000. This project includes also the annotation of the ORFs unique to OG1RF. The OG1RF-unique ORFs are listed in Additional data file 1.Transposon mutations in OG1RF-unique sequencesFollowing the creation of an E. faecalis Tn917 library [12], 6,237 sequences representing the flanking regions of the transposon insertion sites were obtained and compared to the V583 genome by BLASTN. A total of 196 sequences were unique to OG1RF. Thirty-seven of the unique genes contained a transposon insertion. The locations of the transposon insertions are listed in Additional data file 1.Carbohydrate fermentation testsForty-eight E. faecalis isolates, including OG1RF and V583, having different MLST profiles, pulsed field gel electrophoresis types or from various geographical origins, were streaked onto BHI agar and incubated overnight at 37°C. Five to ten colonies of each strain were resuspended in 100 μl of 0.9% saline in a microtiter plate and tested for fermentation using BBL™ Phenol Red Broth Base (Diagnostic Systems, Sparks, MD, USA) supplemented with agar and either 10 mM glucose (positive control), 10 mM dulcitol (negative control), or 10 mM myo-inositol (Sigma, St Louis, MO, USA). Plates were read after incubation at 37°C for 24 h; a yellow halo around the colony was considered positive for fermentation. iolB and iolG2 transposon mutants [12] were also tested.PCRPCR was performed using genomic DNA purified using Bactozol™ (Molecular Research Center, Inc., Cincinnati, OH, USA), as recommended by the manufacturer. Specific PCR primer pairs (Additional data file 4) were used to assess the presence of the OG1RF-unique sequences and for confirmation of flanking DNA regions in common with V583.Competence assaysTo test strains for competence, overnight cultures, grown at 37°C in Todd-Hewitt broth, were diluted in Todd-Hewitt broth to an OD600 nm of 0.05 and then further diluted 10,000-fold in Todd-Hewitt broth to a final volume of 100 ml. After 30 minutes at 37°C, with shaking at 150 rpm, and every hour for 10 h, 0.5 ml samples were removed and 2.5 μg of plasmid DNA were added. The plasmids tested were pAM401 [48] and pMSP3535VA [49]. The samples were incubated for 2 h before plating on BHI or BHI plus antibiotic (chloramphenical 10 μg/ml for pAM401 or kanamycin 2 mg/ml for pMSP3535VA). Following overnight incubation at 37°C, the total numbers of CFU/ml recovered on selective agar for the plasmid were compared to the total number of CFU/ml (plated on BHI agar) for each time point.Biofilm assay and statistical analysisThe biofilm assay was performed as described by Mohamed et al. [68]. Each assay was performed using 16 wells on three occasions. The median was calculated using the 48 OD570 nm readings on data pooled from all experiments and statistical analysis was performed using a non-parametric t-test.Mouse peritonitis modelE. faecalis strains OG1RF and V583 [6] were tested using a previously published method [13]. Mice were injected intraperitoneally with appropriate dilutions of premixed bacteria/sterile rat fecal extract and were observed for five days. Two-fold dilutions of test bacteria in the range 107-109 CFU were used as the inocula for LD50 determination using 6-9 mice per inoculum group. Inocula CFU geometric mean values were obtained and used for LD50 calculation by the method of Reed and Muench [69].UTI model for competition assay and ID50 determinationE. faecalis strains OG1RF and V583 were tested in the UTI model as previously described [16]. For the mixed inoculum experiments, an approximately 1:1 ratio of E. faecalis OG1RF:V583 at approximately 103 CFU each was used. Two independent experiments, using 10 and 11 mice, respectively, were performed and the results were combined. The log10(CFU) of OG1RF and V583 per gram of tissue of each animal (kidney or bladder) from mixed infection were analyzed for significance by the paired t-test. For mono-infection, approximately 103 CFU organisms grown in BHI + 40% horse serum were used for each strain independently and CFU obtained from kidney pairs (nine mice per strain) were analyzed for significance by the unpaired t-test. The minimum detectable limits of recovered bacteria were 101 and 102 CFU/gm of kidney pairs and urinary bladder homogenates, respectively.AbbreviationsATCC, American type culture collection; BHI, brain heart infusion; CC, clonal complex; CFU, colony forming units; CRISPR, comprised of regularly interspaced short palindromic repeats; CSP, competence-stimulating peptide; HK-RR, histidine kinase-response regulator; MGE, mobile genetic element; MSCRAMM, microbial surface component recognizing adhesive matrix molecules; MLST, multilocus sequence typing; ORF, open reading frame; PAI, pathogenicity island.Authors' contributionsGMW, DAG, and BEM designed the study. AB performed much of the post-annotation analysis and non-animal experiments, and wrote the draft of the manuscript. KVS performed the animal experiments. AB, DAG, XQ, JS, SY, AM, KAF, JG, CAA, YS, SRN, MZ, VPP, SC, and SKH annotated the genome. XQ and HJ contributed bioinformatics support. YD, SD-R, CB, HS, GC, GW, DM, LC, and RAG composed the sequencing and finishing team. DAG, BEM, SKH, and GMW assisted in critical review of the manuscript. All authors read and approved the final manuscript.Additional data filesThe following additional data files are available with the online version of this paper. Additional data file 1 is a list of the ORFs unique to OG1RF compared to V583 with their OG1RF locus tag, location in the genome, and definition. Additional data file 2 is a list of genes encoding proteins with a WxL domain in OG1RF and/or V583. Additional data file 3 shows the results of the mouse peritonitis model using OG1RF and V583, with the statistical analysis. Additional data file 4 is a list of the significant primers used in this study.Supplementary MaterialAdditional data file 1OG1RF locus tag, location in the genome, and definition are given.Click here for fileAdditional data file 2Genes encoding proteins with a WxL domain in OG1RF and/or V583.Click here for fileAdditional data file 3Results of the mouse peritonitis model using OG1RF and V583, with the statistical analysis.Click here for fileAdditional data file 4The significant primers used in this study.Click here for file\n\nREFERENCES:\nNo References"
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batch_10/PMC2530874.json

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+{
+    "id": "PMC2530874",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2530874\nAUTHORS: Dong Xia, Sanya J Sanderson, Andrew R Jones, Judith H Prieto, John R Yates, Elizabeth Bromley, Fiona M Tomley, Kalpana Lal, Robert E Sinden, Brian P Brunk, David S Roos, Jonathan M Wastling\n\nABSTRACT:\nA proteomics analysis identifies one third of the predicted Toxoplasma gondii proteins and integrates proteomics and genomics data to refine genome annotation.\n\nBODY:\nBackgroundToxoplasma gondii is an obligate intracellular protozoan parasite that infects a wide range of animals, including humans. It is a member of the phylum Apicomplexa, which includes parasites of considerable clinical relevance, such as Plasmodium, the causative agent of malaria, as well as important veterinary parasites, such as Theileria, Eimeria, Neospora and Cryptosporidium, some of which like Toxoplasma are zoonotic. In common with the other Apicomplexa, T. gondii has a complex life-cycle with multiple life-stages. The asexual cycle can occur in almost any warm-blooded animal and is characterized by the establishment of a chronic infection in which fast dividing invasive tachyzoites differentiate into bradyzoites that persist within the host tissues. Ingestion of bradyzoites via consumption of raw infected meat is an important transmission route of Toxoplasma. By contrast, the sexual cycle, which results in the excretion of infectious oocysts in feces, takes place exclusively in felines.The genome of Toxoplasma has been sequenced, with draft genomes of three strains of Toxoplasma (ME49, GT1, VEG) as well as chromosomes Ia and Ib of the RH strain available via ToxoDB [1]. ToxoDB is a functional genomic database for T. gondii that incorporates sequence and annotation data and is integrated with other genomic-scale data, including community annotation, expressed sequence tags (ESTs) and gene expression data. It is a component site of ApiDB, the Apicomplexan Bioinformatics Resource Center, which provides a common research platform to facilitate data access among this important group of organisms [2]. ToxoDB reflects pioneering efforts that have been made toward the annotation of the Toxoplasma genome. Nevertheless, although the assembly and annotation of the Toxoplasma genome is far in advance of most other eukaryotic pathogens, significant deficiencies still remain; in common with many other genome projects, annotation has thus far not taken into account information provided by global protein expression data and neither have these data been available to the user community in the context of other genome resources.There is now an abundance of transcriptional expression data for Toxoplasma, including expression profiling of the three archetypal lineages of T. gondii. Transcriptional studies have also provided evidence for stage-specific expression via EST libraries, microarray analysis and SAGE (serial analysis of gene expression) [3-6]. Clusters of developmentally regulated genes, dispersed throughout the genome, have been identified that vary in both temporal and relative abundance, some of which may be key to the induction of differentiation [4,6]. Global mRNA analysis indicates that gene expression is highly dynamic and stage-specific rather than constitutive [6]. However, the study of individual proteins has also implicated the involvement of both post-transcriptional and translational control [7-9] and the potential regulation of ribosome expression has also been proposed [10]. Evidence may also point to possible epigenetic control of gene expression, following observations of a strong correlation between regions of histone modification and active promoters [11,12].Until now the study of global gene expression in T. gondii and the use of expression data to inform gene annotation has been almost exclusively confined to transcriptional analyses. Whilst a relatively small number of proteins have been studied in considerable detail, published proteomic expression data are limited to small studies employing two-dimensional electrophoresis (2-DE) separation of tachyzoite proteins [13,14], or to specific analysis of Toxoplasma sub-proteomes that have been implicated in the invasion and establishment of the parasite within the host cell [15-18].This paper reports the first multi-platform global proteome analysis of Toxoplasma tachyzoites resulting in the identification of nearly one-third of the entire predicted proteome of T. gondii and represents a significant advance in our understanding of protein expression in this important pathogen. We describe also the development of a proteomics platform within ToxoDB to act as a public repository for these, and other, proteomic datasets for T. gondii. Our data are now available as a public resource and add a vital hitherto missing dimension to the expression data within ToxoDB. Moreover, the addition of detailed protein expression information within an integrated genomic platform highlights the value of protein expression data not only in interpreting transcriptional data (both ESTs and microarray data), but also provides valuable insights into the annotation of the genome of T. gondii.ResultsTwo-dimensional electrophoresis proteome map of T. gondii tachyzoitesUrea-soluble lysates from cultured T. gondii tachyzoites were resolved using broad (pH 3-10) and narrow (pH 4-7) range 2-DE gels (Figures 1 and 2; Additional data files 1 and 2). The protein identity of individual protein spots was obtained using electrospray mass spectrometry (Additional data files 3 and 4). In total, 1,217 individual protein spots were identified by 2-DE analysis, 783 detected by the pH 3-10 separation and 434 by the pH 4-7 separation. In many instances proteins from separate spots shared the same identity. Examples of clusters of proteins with the same identification are shown boxed in Figures 1 and 2, and these most likely represent isoenzymes, or proteins with post-translational modification. Many gel plugs contained more than one protein and this is represented by overlapping boxes in the figures. Accounting for redundancy between gels and assuming post-translational variants are the products of a single gene, these data represent the expression of 616 non-redundant Toxoplasma genes, of which 547 correspond to release4 gene annotation and 69 are described by alternative gene models or open reading frames (ORFs) that do not correspond to a release4 annotation (discussed further in the 'Genome annotation' section below). Forty release4 genes (which exhibited a range of masses, isoelectric points and functional annotations) were uniquely identified using 2-DE analysis; that is, they were not detected by either the gel liquid chromatography (LC)-linked tandem mass spectrometry (MS/MS) or multidimensional protein identification technology (MudPIT) approaches described in the following sections.Figure 12-DE proteome map (pH 3-10) of T. gondii tachyzoite proteins. Protein spots were visualized using colloidal Coomassie. Spots with the same protein identification are boxed (for detailed numbering, see Additional data file 1). Abbreviations: G1/S phase, G1 to S phase transition protein; Arm RP, armadillo/beta catenin-like repeat containing protein; MLC1, mysosin light chain 1; Sec62, translocation protein Sec62; adenyl cyclase AP, adenyl cyclase associated protein; NPACa, nascent polypeptide associated complex, alpha chain; RBP, RNA binding protein; PKC IC thioredoxin, PKC interacting cousin of thioredoxin; TC tumour protein, translationally controlled tumour protein; BHSP, bradyzoite specific small heat shock protein; Mam33, mitochondrial acidic protein mam33; MSA p30, major surface antigen p30; MDH, malate dehydrogenase; gbp1p protein, gbp1p protein (RNA binding protein); P-serine AT, phosphoserine aminotransferase; inosine-5'-P DH, inosine-5'-monophosphate dehydrogenase; RNA recognition, RNA recognition motif containing protein; nucleolin, nucleolar phosphoprotein (nucleolin), putative; SCR protein, sushi domain-containing protein/SCR repeat-containing protein; nucleosome AP, nucleosome assembly related protein; M2AP, MIC2 associated protein; Rhp23, UV excision repair protein rhp23; PPIase, peptidyl prolyl isomerase; S/T phosphatase 2C, serine/threonine phosphatase 2C; vATPase F, vacuolar ATP synthase subunit F; splicing factor 3b/10, splicing factor 3b subunit 10; 40S RP S12, 40S ribosomal protein S12; eTIF1a, eukaryote translation initiation factor 1 alpha; eTIF3d, eukaryote translation initiation factor 3 delta subunit; PPIPK, phosphatidylinositol-4-phosphate 5-kinase; LDH, lactate dehydrogenase; RACK, receptor for activated C kinase; LGL, lactoylglutathione lyase; Ca2+ BP, membrane associated calcium binding protein; IPP2A, inhibitor 1 or protein phosphatase type 2A; HPPK/DHPS, hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase; RNA BP, RNA binding motif protein; La protein, La domain containing protein; Pfs77r, pfs77 related protein; P-protein, phosphoprotein; PPI/WD, protein with peptidylprolyl isomerase domain and WD repeat; dUTP hydrolase, deoxyuridine 5'-triphosphate nucleotidohydrolase; PRE3, proteasome component PRE3 precursor; 10 kDa HSP mito, mitochondrial heat shock protein; PPIase NIMA, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1; CEP52 fusion protein, ubiquitin/ribosomal protein CEP52 fusion protein.Figure 22-DE proteome map (pH 4-7) of T. gondii tachyzoite proteins. Protein spots were visualized using colloidal Coomassie. Spots with the same protein identification are boxed (for detailed numbering, see Additional data file 2). Abbreviations (also refer to Figure 1): PSAT, phosphoserine amino transferase; IF4E, translation initiation factor 4E; BCDC E1, branched-chain alpha-keto acid dehydrogenase; SOD, superoxide dismutase; OGDC E2, dihydrolipoamide succinyltransferase component of 2-oxoglutaratedehydrogenase complex; EGF1b, elongation factor 1 beta; ubiquitin-E2, ubiquitin-conjugating enzyme E2; F-1,6 bisP aldolase, fructose, 1,6 bis phosphate aldolase; PGK, phosphoglycerate kinase; F1,6 b Pase, fructose 1,6 bis phosphatase; U5 snRNP, U5 snRNP-specific 40 kDa protein (hPrp8-binding); Dihydrolipoyl DH, Dihydrolipoyl dehydrogenase, third enzyme of PDC, OGDC, BCDC.T. gondii tachyzoite proteome analysis by one-dimensional electrophoresis gel LC MS/MSWhole tachyzoite protein, solubilized in SDS, was resolved using a large format one-dimensional electrophoresis (1-DE) gel (Figure 3). We excised 129 contiguous gel slices from the entire length of the resolving gel and each gel slice was submitted to LC-MS/MS. This approach combines the resolving power of SDS gel-based protein separation with that of the liquid chromatography separation coupled on-line to the mass spectrometer and resulted in the generation of large, high quality datasets of SDS-soluble proteins. An average of 20 proteins was identified from each 1 mm gel slice and the complete dataset comprising 2,778 individual protein identifications is shown in Additional data file 5. A further 1-DE experiment, using prior Tris solubilization, led to the identification of 82 additional release4 genes and 9 alternative gene models (Additional data files 6 and 7). Some proteins were identified in multiple gel slices again, likely due to isozymes or post-translational modifications. When redundancy between proteins with the same identification was removed, 1,012 individual gene products (939 release4 and 73 alternative gene models) were identified from T. gondii tachyzoites by gel LC-MS/MS analysis (Additional data files 8 and 9).Figure 3Tachyzoite proteins resolved for 1-DE gel LC-MS/MS. SDS-soluble proteins from 1.1 × 108 tachyzoites were resolved on a 12% (w/v) acrylamide gel under denaturing conditions as follows: protein standards (lane 1); T. gondii soluble protein (lane 3). Proteins were visualized using colloidal Coomassie stain.MudPIT analysis of T. gondii tachyzoitesWhole tachyzoite protein was partitioned into Tris-soluble and Tris-insoluble fractions, and each processed for MudPIT analysis; this resulted in 1,300 and 2,328 protein identifications, respectively, and a total non-redundant dataset comprising 2,409 proteins, which comprises 2,121 release4 and 288 alternative gene models (Additional data files 10 and 11). Of the release4 genes identified, 15.3% were identified uniquely in the Tris-soluble fraction and 48.0% were identified uniquely in the Tris-insoluble fraction.When the results using all three proteomic platforms were combined, a total of 2,252 non-redundant release4 protein identifications were obtained from the tachyzoite stage of the parasite. This represents expression from approximately 29% of the total number of currently predicted release4 genes. Figure 4 illustrates the degree of overlap between the datasets derived using each of the three proteomic platforms. MudPIT generated the largest number of identifications; however, a number of proteins were uniquely identified using the gel-based approaches (59 for 1-DE; 40 for 2-DE). Other studies have also highlighted the benefits of a multi-platform proteomic approach and the advantages and disadvantages of each platform have been discussed extensively elsewhere [19]. Notably, the gel-based proteomic platforms detected, on average, more peptides per protein identification than MudPIT. Overall across all platforms, only approximately 6% of the 2,252 proteins identified were based on single peptide evidence; this represents a relatively low proportion compared to other apicomplexan proteomic studies [19-21] and is probably accounted for partly by the extensive data from gel-based proteomics in addition to the MudPIT analysis. In addition to the release4 genes, 394 non-redundant alternative gene models and ORFs were also identified from the entire dataset. These data represent sets of peptides that map more comprehensively to alternative models and ORFs than the release4 gene models, and have considerable implications for genome annotation, as discussed below.Figure 4The tachyzoite expressed proteome: comparison of proteome strategies. Venn diagram showing the numbers of unique and shared non-redundant release4 gene identifications obtained from each of the three proteomics platforms.Functional analyses and key pathways of the tachyzoite proteomeEach individual protein detected by proteomics was submitted to the motif prediction algorithms SignalP [22] and TMHMM [23] and also to subcellular localization prediction programs, for example, PATS (apicoplast) [24], PlasMit (mitochondrion) [25], WoLF PSORT (general) [26] and Gene Ontology (GO) cellular component prediction downloaded from ToxoDB. Toxoplasma genome predictions suggest that 11% of proteins contain a signal peptide and 18% contain transmembrane domains (information available at ToxoDB). Virtually identical proportions were detected in this study in the expressed proteome of tachyzoites (10% and 18%, respectively). Analysis of the 394 alternative gene models and ORFs gave closely similar proportions (results not shown). This represents expression of more than one-quarter of the predicted numbers of membrane and secreted proteins within one life-cycle stage of the parasite. Assuming non-biased sampling, these results imply no enrichment for membrane proteins in tachyzoites. Similar proportions of signal peptide and transmembrane containing proteins were observed in the expressed proteome of Plasmodium falciparum [20]. The Toxoplasma proteins showed a wide distribution of sub-cellular localizations, demonstrating broad sampling, with cytoplasmic, nuclear and mitochondrial locations well represented (Figure 5a; Additional data file 12). Many proteins were also potentially involved in secretory pathways and were assigned to the endoplasmic reticulum-Golgi, the plasma membrane and extracellular locations.Figure 5Subcellular localisation and functional categorization of the expressed tachyzoite proteome. The numbers correspond to the total number of identified proteins in each category. (a) Protein subcellular localization information was first assigned according to gene descriptions and GO annotation provided by ToxoDB. When no information was available, protein sequences were submitted to PATS, PlasMit and WoLF PSORT. The combined results were manually assessed to obtain subcellular localization predictions. A detailed list of proteins in each subcellular localization to accompany this figure is provided in Additional data file 12. (b) Functional categorization was constructed using the GO classifications listed on ToxoDB for each release4 gene, which were then assigned to specific MIPS categories within the FunCatDB functional catalogue. Genes without a GO classification were assigned a putative MIPS category using additional information provided by Blast, Pfam domain alignments, InterPro and from independent literature searches. Notes: protein fate includes protein folding, modification and destination. A detailed list of proteins in each functional category to accompany this figure is provided in Additional data file 13.The functional analysis of the expressed proteome presented in Figure 5b (see also Additional data file 13) was constructed using the GO classifications listed on ToxoDB, which are largely based on bioinformatics interpretation. Each release4 gene was then assigned to a specific Munich Information Centre for Protein Identification (MIPS) category within the FunCatDB functional catalogue [27]. Some genes are without a GO classification and were assigned a putative MIPS category using additional information provided by Blast similarities, Pfam domain alignments [28], InterPro [29], orthologs, Toxoplasma paralogs, and from independent literature searches. Functional categories that are highly represented are metabolism, protein fate, protein synthesis, cellular transport, transcription and proteins with binding functions. A large proportion (36%) of the proteins have 'unknown function', indicating the difficulty of obtaining functional information using sequence similarity methods alone. Functional assignments were also constructed for hits to alternative gene models and ORFs, revealing similar relative proportions of functional categories, except for a larger proportion (70%) of proteins with unknown function, presumably due to the sequences being atypical, or incompletely predicted (Additional data file 14). The implications of the functional categories discovered are examined in the Discussion.Tachyzoites are thought to rely upon both glycolysis and the tricarboxylic acid cycle, unlike the bradyzoites, which are thought to be largely dependent upon glycolysis [7]. Virtually every component of the glycolysis/gluconeogenesis pathway predicted for Toxoplasma was identified as being expressed in tachyzoites by proteomic analysis, as illustrated in Figure 6. Additionally, considerable coverage of the oxidative phosphorylation and tricarboxylic acid cycle pathways was also identified from the expressed proteome dataset (data not shown; see ToxoDB for further details). Several enzymes of the glycolytic pathway have been shown to be modulated during differentiation [6,7], with some showing stage-specific isoforms, such as enolase and lactate dehydrogenase [8]. The level of mRNA expression does not always mirror that of the expressed protein, indicating a degree of translational control or changes in mRNA stability [8]. However, it should be noted that detecting low levels of protein can be problematic. One example is glucose-6-phosphate isomerase (76.m00001). Western analysis detected expressed protein in bradyzoites but not tachyzoites despite the presence of abundant mRNA transcripts in both stages [30]. However, glucose-6-phosphate isomerase was successfully detected in tachyzoites in this whole cell proteome analysis (Additional data file 5, gel slices 40-42), again illustrating the sensitivity of our proteome approach.Figure 6Metabolic pathway coverage: glycolysis/gluconeogenesis. Component enzymes of the glycolysis/gluconeogenesis pathways predicted to be present in Toxoplasma from genome analysis are colored. Virtually every component of the glycolysis/gluconeogenesis pathway predicted for Toxoplasma was identified as being expressed in tachyzoites by proteomic analysis. Green and blue indicate genes for which expression has been confirmed in tachyzoites in this study by mass spectrometric data; blue also signifies genes for which post-translational modification is likely as indicated by the evidence from two-dimensional gels. Red indicates genes for which expression of predicted components has not been confirmed in this study. Coverage of key metabolic pathway component proteins was determined using the Metabolic Pathway Reconstruction for T. gondii available on the KEGG Pathway site accessed via ToxoDB [53].Comparison with EST expression dataFigure 7a illustrates the degree of correlation between release4 genes for which EST expression data are available and genes for which the total proteome dataset identified in this study has provided evidence of expression. By including all the tachyzoite and bradyzoite cDNA evidence from RH, ME49, VEG, CAST, COUG and MAS strains (available at ToxoDB), most (91%) of the proteins found in this study were corroborated by EST data. Approximately half of these were confirmed in both bradyzoite and tachyzoite stages by EST analysis, suggesting that many of the proteins may have common, house-keeping functions. Although the EST coverage of the total number of release4 genes listed at ToxoDB is relatively high (68% for tachyzoite ESTs alone), for 266 release4 genes detected in this study using proteomics there was no corresponding tachyzoite EST evidence, apparently reflecting inadequacies in the coverage of the EST data. The distribution of cellular functions amongst these 266 expressed proteins is representative of the entire proteome dataset, indicating that EST evidence is lacking for many different proteins and not specific for a particular type or category of function (data not shown).Figure 7The tachyzoite expressed proteome: comparison with EST and microarray expression data. A comparison of the expressed proteome of tachyzoites with EST and microarray data reveals discrepancies between protein and transcriptional data. (a) Venn diagram comparing the correlation between the number of non-redundant release4 genes detected by EST expression from T. gondii tachyzoite and bradyzoites (available from ToxoDB) and those detected by this proteome study. The number of genes unique to each intersection is indicated. (b) Venn diagrams comparing the correlation between release4 genes obtained by this proteome study and those detected by microarray analysis of RH strain tachyzoites, including those genes with expression of ≥ 25 and ≥ 50 percentiles. (c) Bar chart showing the number of release4 genes also detected by proteomics for each of the four percentile ranges, 0-24%, 25-49%, 50-74%, 75-100%, determined by microarray analysis.Conversely, comparison of RH strain-specific tachyzoite ESTs with the proteome dataset revealed that 57% of genes for which there was EST transcript evidence were not corroborated by the detection of expressed protein in this study. This is likely to be explained by a number of contributing factors, including the difficulty in detecting low copy number, transient and unstable proteins. It is also possible that a small number of non-coding ESTs are present in the database for which no protein product would be expected.Comparison with microarray dataMicroarray analysis of the RH strain of T. gondii has been performed previously (data available through ToxoDB; A Bahl and DS Roos unpublished). The analysis provides extensive coverage of the genome (99.5% of release4 genes were assayed), and the results have been cross-referenced with the proteins identified. As it is difficult to determine the correct signal:noise ratio above which mRNA levels can be considered to be indicative of a gene being switched on (all genes represented on the array exhibit some signal, yet not all are expressed), the microarray results were divided into quartiles of mRNA expression level for the purposes of this comparison. Those genes in the bottom 25% were described as zero detectable mRNA above baseline, and alternatively those in the bottom 50% were described as having zero or low detectable mRNA level. The Venn diagrams in Figure 7b illustrate the degree of overlap between release4 genes, for which ≥ 25 percentile and ≥ 50 percentile mRNA expression was detected by microarray analysis, and the genes identified by our proteomic study. The results illustrate that some genes with zero or low mRNA can still be identified in a proteome study (204 proteins matching the < 25% group and 632 proteins matching the < 50% group). The detection of these proteins is intriguing and there may be several possible explanations. For example, these proteins may be highly stable and do not require new transcription for the protein to be detected, or perhaps substantial quantities of protein can be produced from very low mRNA. Three examples from this group are: 'bi-functional aminoacyl-tRNA synthetase, putative/prolyl-tRNA synthetase, putative' (38.m00021, 254 peptide hits), 'clathrin heavy chain, putative' (80.m02298, 148 peptide hits) and 'KH domain-containing protein' (35.m00901, 136 peptide hits). The high number of peptide hits demonstrates that these proteins are clearly present in high copy number yet have little or no detectable mRNA; such proteins are interesting candidates for understanding the relationship between mRNA and protein abundance levels in Toxoplasma.Figure 7c displays the comparison of the number of proteins identified matching each quartile of genes, according to mRNA expression level. There is a general trend for more proteins to have been detected for genes with higher mRNA expression levels (from the top quartile, 972 proteins have been detected, and only 204 have been detected from the bottom quartile), indicating, as expected, that there is some correlation between mRNA abundance and protein abundance.Genome annotation and generation of a public proteome interface for ToxoplasmaThe mass spectrometry data in this study were searched against a database containing the current set of predicted proteins from ToxoDB (referred to here as release4), predicted proteins derived from alternative gene models (GLEAN, TigrScan, TwinScan and Glimmer), ESTs and a translation of all six ORFs (see Materials and methods). As such, the proteome data can provide evidence that an alternative gene model is the correct prediction, or that a gene has not been predicted at all in the genome.The release4 annotation available in ToxoDB release 4.2 was provided by the Toxoplasma Genome Sequencing Project. The proteome data have been aligned with release4 gene annotations where possible for identified peptide sequences that exactly match a protein predicted in the release4 set. These peptides can be viewed in relation to the predicted protein and the genomic region from which the sequence is predicted to have been produced. The peptide identifications can be viewed in the ToxoDB genome browser GBrowse by selecting the option 'Mass Spec Peptides (Wastling, et al.)'. This dataset comprises 2,252 release4 genes. In addition, identified peptides that are more likely to have arisen from a translation of an alternative gene model have been aligned, and can be viewed in GBrowse by selecting the option 'Mass Spec Peptides (Alternative Models)'.For the majority of annotated genes, integration of the expressed peptide data has provided direct confirmation of the correct prediction of ORFs and positioning of exon-intron boundaries, including a large number of hitherto 'hypothetical proteins'. The further significance and importance of this corroboratory evidence become more apparent when considering the minority of cases where the peptide expression data are in conflict with the gene prediction algorithms. Approximately 15% of the complete proteome dataset consists of peptide hits to regions of the scaffold where there are discrepancies with the new gene annotation and peptides mapped more convincingly to alternative gene models or ORFs (that is, 394 protein coding sequences). Of the 394 alternative gene models and ORFs detected, most are described as 'hypothetical' with minimal information available and were detected using MudPIT analysis. These hits can be viewed at ToxoDB using the queries and tools option that guides the user to a main menu page from which gene expression confirmation via mass spectrometry can be accessed. The option of refining the search to a single or combination of proteomic approaches, and of searching either annotated genes or ORFs, is available. By adopting the GBrowse viewing option, the user can examine in detail individual ORFs and the integrated peptide sequence data.An example is illustrated in Figure 8 of a region of the scaffold where peptide evidence supports the presence of an expressed ORF but the new prediction algorithm has not assigned a gene in the corresponding region. Eleven peptides map to TgGlmHMM_3355 and TgTigrScan_5280 but the release4 annotation does not predict an exon in this region. Additional peptides in this region map to exons of the neighboring gene 46m.02877; however, these peptides could also be assigned to the coding sequence of TgGlmHMM_3355 and/or TgTigrScan_5280. In this case, the peptide evidence appears to indicate that gene 46m.02877 could have an incorrect start methionine and be missing an amino-terminal exon.Figure 8Peptide evidence indicating an ORF where release4 annotation does not predict an ORF. The position of ORF X-3-4725402-4726856 in the genome scaffold is indicated by a red line on the grey track at the top of the figure and this region is expanded below, the red triangle demarking the ORF length. Different gene annotation models are presented one above the other bellow the scaffold. Predicted exons are indicated as blue boxes, linked by zigzag lines to indicate the position of exon/intron boundaries. The predicted sequence for TgGlmHMM_3355 is shown as an insert; sequence for which there is matching peptide evidence is shown in red. The peptide that spans an intron-exon boundary is shown in purple. Peptides aligning with this region are shown in yellow and the detailed MS information for one is shown, including the predicted sequence. Peptides that align with the release4 or alternative gene annotations are indicated on different lines. ESTs are shown as dark blue or brown boxes.In other cases, peptide identifications are able to identify errors in the predicted reading frame or strand orientation as illustrated in Figure 9. Here 12 peptides derived from 35 individual spectra originating from both 1-DE and MudPIT approaches provided matching hits to TgGlmHMM_1717, TgTwinScan_4462 and TgGLEAN_7850, whereas the new gene prediction algorithm (assigned 50.m05694) is predicted to lie on the opposite strand and TgTigrScan_8273 uses a different reading frame. The various algorithms also differ in the predictions of the length and number of exons, although peptide evidence supports a single exon. In this example, the peptide expression data have provided supporting evidence for the correct reading frame and the large number of peptide hits to one region only indicates that the gene is likely to comprise a single exon.Figure 9Peptide evidence indicating alternative frame shift. The position of ORF XII-4-5562689-5562144 in the genome scaffold is indicated by a red line on the grey track at the top of the figure and this region is expanded below, the red triangle demarking the ORF length. Predicted exons are indicated as red shaded boxes, linked by zigzag lines to indicate the position of exon/intron boundaries. Peptides aligning with this region are shown in yellow. The gene of interest with the release4 annotation (50.m05694) is highlighted in blue. Predicted sequences for this gene and the ORF and TgGlmHMM_1717 are shown as inserts. Sequence for which there is matching peptide evidence is shown in red. TgGlmHMM_1717 comprises several exons and the complete sequence is not given; the start methionine is shown in green. Mass spectrometric evidence for one peptide sequence derived by the 1-DE approach is shown.Other discrepancies involving the positioning of the exon-intron boundaries exist and, in some cases, the alternative gene annotation models such as TgGlmHMM, TgTigrScan, TgTwinScan and TgGLEAN correlate more closely with the co-ordinates of the peptide data. In Figure 10, 12 peptides from MudPIT analysis map to a region of the scaffold (X: 3917326-3920484) that is annotated with gene 28.m00300, comprising two exons. Five of the twelve peptides match the second exon of gene 28.m00300. While it appears that peptides match the scaffold in the region of 28.m00300 exon 1, these peptides have been predicted from a different frame translation. Of further note is that one peptide maps to the predicted intron region of gene 28.m00300. Alternative gene models vary considerably in this region of the scaffold in both the number and positioning of the exons and all 12 peptides only appear in TgGlmHMM_2666, which does not have an intron at this location, providing evidence that this model is most likely to be correct.Figure 10Peptide evidence indicating alternative exon positioning and sequence annotation. The position of ORF X-1-3917326-3920484 in the genome scaffold is indicated by a red line on the grey track at the top of the figure and this region is expanded below, the red triangle demarking the ORF length. Predicted exons are indicated as blue boxes, linked by zigzag lines to indicate the position of exon/intron boundaries. Gene 28.m00300 is shown with two exons. ESTs are shown as dark blue or brown boxes. Peptides aligning with this region are shown in yellow. The predicted sequence for ORF X-1-3917326-3920484 is shown as an insert and sequence that matches exon 2 of gene 28.m00300 is shown in blue. Sequence for which there is matching peptide evidence is shown in red. Purple lettering indicates the positioning of the 'intron-located' peptide, mass spectrometric evidence for which is shown in the right hand insert.An important use of peptide identification is to confirm that intron-exon (splice) boundaries have been correctly predicted; these are notoriously difficult to predict accurately in genome sequence using informatics approaches alone. If a peptide sequence spans an intron, matching regions from the splice donor and acceptor of two exons, this provides strong evidence that splicing has been correctly predicted for these exons. In total, our study identified 2,477 intron spanning peptides in the official release4 annotation, providing supporting evidence that these splice sites have been correctly predicted. In addition, peptides aligning across 421 splice boundaries predicted from alternative gene models only have been identified. This number is highly significant, as the identifications provide strong evidence that the alternative gene model is correct for this region, allowing the genome annotation to be improved. One example of a peptide spanning an intron is shown in Figure 8, where peptides have been identified that span an intron between exons predicted by TwinScan and Glimmer only.DiscussionDraft genomes now exist for the majority of clinically important protozoa, including most Apicomplexa. Providing an accurate interpretation of gene annotation and expression from these genomes is essential to understanding the biology of host-pathogen interactions and in gaining a better understanding of the relationship between gene transcription and protein expression. Of particular importance is an appreciation of the limitations that transcriptional data alone place on our interpretation of how pathogens respond as they develop through different life-stages, or during key processes such as invasion and establishment within their hosts. Such an observation has potentially huge implications for expression profiling and for the reliance on microarray data to describe changes in gene expression. In this paper we describe how global proteomic data for T. gondii provides important insights into both genome annotation and gene expression in this model apicomplexan parasite.Proteomic data enable us to understand what is actually expressed, as opposed to what might be, or has the potential to be, expressed in an organism. In general, the functional characterization and protein localization profile detected in T. gondii in this study fits well with that of the rapidly dividing and invasive tachyzoites, which would be expected to be highly metabolically active, with gene expression, protein synthesis, remodeling and degradation all necessary processes involved in active parasite cell division and required for successful host cell invasion. A similar profile was recently obtained for the expressed proteome of the invasive form of Cryptosporidium [19]. Penetration and maintenance within the host cell would require expression of many apical organelle proteins involved in invasion (category: cell rescue, defense and virulence), as has been observed for the invasive stages of Plasmodium and Cryptosporidium [19,20,31]. In agreement, 44 proteins were assigned to an apical organelle location in Figure 5a. Recent work has also shown the recruitment of host endoplasmic reticulum, mitochondria and networks of intimately proximal microtubules facilitating active transport of host nutrients to the parasite [32-35]. Notably, proteins involved in cellular transport are well represented, with more than 200 expressed in this life cycle stage. A significant proportion of proteins falls into the broad category 'proteins with binding functions', including proteins involved in the cytoskeleton that are also required for motility, an important function during invasion. Many proteins were also detected that would be expected to be expressed at low or temporal levels within the cell, such as those involved in cell cycle control (641.m01576, 38.m00005) or signal transduction (65.m01199, 59.m06067, 55.m04992, 49.m05708, 50.m05649). This suggests that the sensitivity of our proteomic analyses was high.Perhaps most notable were the large number of proteins (36%) for which no information is available and these proteins are listed as unclassified. A similarly large proportion (39%) of proteins with unknown function were detected in just one life cycle stage (the sporozoites) of Cryptosporidium by proteomic analysis [19] and in the proteome of four life cycle stages of P. falciparum (that is, 51%) [20]. More than half the predicted genes of Toxoplasma are annotated as 'hypothetical' in the genome. In this analysis, around 800 genes annotated as 'hypothetical protein' were identified, allowing these annotations to be updated to 'confirmed protein'. Functional analysis was also carried out on the 394 alternative gene models and ORFs and revealed a far greater proportion of proteins for which a functional assignment could not be determined (70% compared to 36%). This result reflects the limited annotation available for alternative gene models and ORFs, partially due to the short length of many of these sequences and difficulties obtaining functional information by sequence similarity search if the predicted ORF or alternative gene models do not closely resemble the correct gene sequence.Toxoplasma has a complex life cycle comprising four additional life cycle stages not studied here: the infective sporozoite, two sexual stages and the encysted bradyzoite. Many house-keeping proteins will be common to all stages, although the proportion of shared proteins is not currently known. In this analysis, approximately one-third of the predicted number of release4 genes were detected in the proteome of the tachyzoite, although it is important to remember that these predicted genes will include stage-specific genes not expressed in the tachyzoite stage, so the actual proportion of proteins detected compared to those expected is likely to be considerably higher, although how much higher is impossible to determine at this stage. Whole cell proteome analysis of the related apicomplexan parasite, Cryptosporidium parvum, indicated expression of a similar proportion of the genome from the infective sporozoite stage [19], and this parasite also exhibits multiple life cycle stages. Whether the protein set detected is close to the complete proteome of the life cycle stage or limited by the detection levels of the mass spectrometric techniques is not yet clear. Previous microarray analysis of sporozoites, gametocytes and blood stage life cycle stages of Plasmodium indicated 35% of genes were shared [36] whereas this figure decreased to 6% at the proteome level [20,37]. It is likely that some of this discrepancy results from technical limitations associated with detecting low abundance proteins, although it is possible that post-transcriptional regulation also plays a role. In Toxoplasma, analysis of 568 EST assemblies from three life cycle stages, tachyzoites, bradyzoites and oocysts, indicated 16% of genes are stage-specific and, hence, that a large proportion of the genes is shared [5]. A similar figure of 18% was obtained via SAGE analysis [6].The comparison of the detected proteome with microarray results also reveals some interesting discrepancies. Of the least abundant 25% mRNA values, which would usually be described as no measurable mRNA signal above baseline, 204 proteins are detected. In contrast, of the genes with most abundant mRNA (top 25%, approximately 1,900 genes), only half of these are detected by proteome analysis. The most abundant proteins are likely to have been sampled preferentially in this analysis, and as such, we can hypothesize that many of the genes expressing high mRNA levels do not exhibit similarly high abundances of protein product. Without an in-depth absolute quantitative study of the complete Toxoplasma proteome, which is highly challenging with current technology, these results should not be over-interpreted. However, it appears that there is a considerable degree of control that regulates the level of protein abundance, independent of the rate of transcription in tachyzoites.Our proteome data have been integrated and aligned with the genome sequence at ToxoDB. The interface provided enables visual inspection of peptides matched to the most current (in this case 'release4') gene models, as well as to alternative gene models and ORFs. The facility to visualize and query peptide data, in tandem with EST and microarray data, allows users of ToxoDB to place confidence in particular gene assignments and to explore those genes that are expressed in tachyzoites. As demonstrated above, the proteome data will enable continued improvement in gene models through the confirmation of the correct reading frame and intron-exon boundaries. More fundamentally, the proteome analysis raises several issues in relation to the correct determination of gene models. Many gene prediction algorithms work on the basis of sequence similarity to cDNA or protein sequence databases, EST sequences or other genome sequences (where conserved regions are more likely to correspond to genes). As such, gene finders are relatively successful at identifying 'typical' genes that are similar to gene structures previously observed in other organisms. However, where genes are atypical in structure, or have no EST data, gene finding algorithms may miss such sequences altogether. Large-scale proteome scans are able to contribute significantly in this area, by demonstrating peptide hits to regions of the genome where genes have only been weakly predicted or missed completely. Others have recently also recognized the value of so-called 'proteogenomic annotation' of genomes [38-42]. As more proteome data are produced, and querying algorithms improve, it is likely that the majority of protein-coding genes expressed in Toxoplasma will be confirmed by mass spectrometry based evidence.ConclusionThis study represents an unprecedented integration of proteomic and genomic data for Toxoplasma, which we suggest might serve as a model well beyond this present field. As well as providing novel information on the functional aspects of the proteome, our data demonstrate how proteomics can inform gene predictions and help discover new genes. Moreover, the data reveal some surprising, but potentially highly significant, discrepancies between protein expression and transcript expression data as assessed by both EST analysis and microarrays. We believe that this has important implications for how we interpret transcriptional expression data in the Apicomplexa, such as that derived from microarray experiments, and points to the fact that determining both absolute protein expression and post-translational events will be a key factor in gaining a more complete understanding of the biology of these pathogenic organisms.Materials and methodsChemicals and materialsChemicals were AnalaR or HPLC grade and from VWR (Poole, UK) except: amidosulphobetaine-14 (ASB-14; Calbiochem, Nottingham, UK); deoxycholate (Sigma-Aldrich, Steinheim, Germany); iodoacetamide (Sigma-Aldrich); Invitrosol (Invitrogen, Carlsbad, CA, USA); Mini complete protease inhibitor cocktail (Roche, Penzberg, Germany); bovine pancreas sequencing grade trypsin (Roche); thiourea (Sigma-Aldrich); TCEP (tris (2-carboxyethyl) phosphine hydrochloride (Pierce, Rockford, IL, USA); 2-DE consumables (Amersham Biosciences, Little Chalfont, UK).Parasite cultureTachyzoites of T. gondii strain RH were maintained in confluent layers of Vero cells (ECACC, Salisbury, UK). T. gondii tachyzoites were harvested 3 or 4 days post-infection as previously described [13].One-dimensional PAGE analysisA pellet of 1.1 × 108 tachyzoites (approximately 220 μg) was solubilized in 40 μl of 100 mM Tris/HCl pH 6.8, 10% (v/v) glycerol, 4% (w/v) SDS, 0.01% (w/v) Bromophenol Blue, 200 mM dithiothreitol (DTT), with three cycles of 5 minutes at 90°C and 2 minutes vortexing, then spun at 16,000 g for 3 minutes. The supernatant was run on a 16 cm 12% (v/v) acrylamide gel using the denaturing Tris-glycine method of Laemmli [43], at 16 mA for 30 minutes and 24 mA for 6-7 h at 15°C. The gel was stained with colloidal Coomassie blue, the lane cut into 129 slices of < 1 mm thickness and each digested with trypsin. For the Tris-fractionated sample, a pellet of 9.85 × 107 tachyzoites was solubilized on ice for 1 h in 50 μl of 100 mM Tris/HCl pH 8.5 and vortexed every 10 minutes. Three cycles of freeze-thaw using liquid nitrogen, and 2 minutes of vortexing followed, and the sample spun at 16,000 g at 4°C for 30 minutes to partition Tris-soluble protein (supernatant) from Tris-insoluble protein (pellet). The latter was further solubilized in 50 μl of 2% (v/v) SDS, 100 mM DTT using three cycles of 5 minutes at 90°C and 2 minutes vortexing, with a final spin at 16,000 g for 15 minutes. An aliquot of 20 μl of 100 mM Tris/HCl pH 6.8, 10% (v/v) glycerol, 4% (w/v) SDS, 0.01% (w/v) Bromophenol Blue, 200 mM DTT was added to 30 μl of Tris-insoluble protein (approximately 130 μg), and to 30 μl of Tris-soluble protein (approximately 120 μg) and resolved on a 12% (w/v) acrylamide gel as described above. Twenty-five gel slices were excised from a region of the gel deemed to exhibit maximum density and variation in protein banding.Two-dimensional PAGE analysisFrozen pellets of T. gondii tachyzoites were solubilized in 7 M urea, 2 M thiourea, 4% (w/v) Chaps, 2% (w/v) ASB14, 20 mM Tris base, 60 mM DTT, 1 mM EDTA, 1 × Mini Complete protease cocktail inhibitor, 0.5% (v/v) immobilized pH gradient (IPG) strips buffer (pH 4-7 linear gradient, 1 × 108 tachyzoites, approximately 200 μg; pH3-10 non-linear gradient, 2.58 × 108 tachyzoites, approximately 516 μg). The samples were incubated at room temperature for 4-5 h with a vigorous vortex every half an hour and spun at 16,000 g for 5 minutes. The supernatants were made to a final volume of 450 μl with 8 M urea, 2% (w/v) CHAPS (3- [(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate), 0.002% (w/v) Bromophenol Blue, 40 mM DTT, supplemented with 0.5% (v/v) pH 3-10 NL or pH 4-7 L IPG buffer and used to rehydrate 24 cm Immobiline IPG strips for a minimum of 10 h at room temperature. The rehydrated strips were placed on an Ettan™ IPGphor II™ with a loading manifold (GE Healthcare, Bucks, UK) and isoelectric focusing (IEF) was run at 20°C, 75 μA per strip as follows: stepped voltage, 500 V for 2 h; gradient voltage, 1,000 V over 8 h; gradient voltage, 10,000 V over 3 h; stepped voltage, 10,000 V for 4 h and 15 minutes (approximately 65, 000 Volt hours). The IPG strips were equilibrated for 15 minutes each in 6 M urea, 50 mM Tris/HCL pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% (w/v) Bromophenol Blue supplemented with 1% (w/v) DTT, then with 2.5% (w/v) iodoacetamide and mounted on DALT 12.5% (w/v) pre-cast 24 cm acrylamide gels resolved using an Ettan DALT™ 6-MultiTemp III apparatus and buffering kit (Amersham Biosciences). Gels were run at 20°C, 3 W for 0.5 hour and 17 W per gel thereafter.Colloidal Coomassie stainingGels were fixed in 40% (v/v) ethanol, 10% (v/v) acetic acid overnight at room temperature, rinsed in distilled deionized water, stained for 5 days with colloidal Coomassie stain (20% (v/v) methanol, 0.08% (w/v) CBB G250, 0.8% (v/v) phosphoric acid, 8% (w/v) ammonium sulfate), rinsed in distilled deionized water and stored in 1% (v/v) acetic acid at 4°C.In-gel tryptic digestionGel plugs/slices were destained at 37°C using 50 mM ammonium bicarbonate/50% acetonitrile. One-dimensional gel slices were incubated at 37°C with 10 mM DTT/100 mM ammonium bicarbonate for 30 minutes, then 100 mM iodoacetamide/55 mM ammonium bicarbonate for 1 h in the dark. Gel plugs/slices were dehydrated with 100% (v/v) acetonitrile at 37°C and rehydrated at 37°C with 10 μl of 10 ng/μl sequencing grade trypsin in 25 mM ammonium bicarbonate. After 1 h, 25 mM ammonium bicarbonate was added to cover the gel pieces, which were left at 37°C overnight. The reaction was stopped with 2 μl of 2.6 M formic acid and the samples stored at -20°C.Tandem mass spectrometry (LC-MS/MS)LC-MS/MS was performed on an LTQ ion-trap mass spectrometer (Thermo-Electron, Hemel Hempstead, UK) coupled on-line to a Dionex Ultimate 3000 (Dionex Company, Amsterdam, The Netherlands) HPLC system equipped with a nano pepMap100 C18 RP column (75 μm; 3 μm, 100 Angstroms) equilibrated in 98.9% water/2% acetonitrile/0.1% (v/v) formic acid at 300 nl/minute. Tryptic peptides were desalted on a C18 TRAP, and resolved with a linear gradient of 0-50% (v/v) acetonitrile/0.1% (v/v) formic acid over 30 minutes, followed by 80% (v/v) acetonitrile/0.1% (v/v) formic acid for 5 minutes. Ionized peptides were analyzed using the 'triple play' mode (0-106 m/z, global and Msx), consisting initially of a survey (MS) spectrum from which the three most abundant ions were determined (threshold = 200-500 TIC [total ion chromatogram]). The charge state of each ion was assigned from the C13 isotope envelope 'zoom scan', fragmented (collision energy 35% for 30 ms) and subjected to a MS/MS scan. The LTQ was tuned using a 500 fmol/μl solution of glufibrinopeptide (m/z 785.8, [M+2H]2+). The resulting MS/MS spectra were submitted to TurboSequest Bioworks version 3.1 (Thermo Fisher Scientific Inc., Waltham, MA, USA) (threshold cut-off 0-1000; group scan default 100; minimum group count 1; minimum ion count 15; peptide tolerance 1.5), the individual spectra (dta files) merged into an mgf file and submitted to Mascot (Matrix Science, London, UK) and searched against a locally mounted Toxoplasma genome database comprising ORFs > 50 amino acids; clustered ESTs; whole genome shotgun (10×); TwinScan, TigrScan and GlimmerHMM protein predictions; and T. gondii annotated proteins_ToxoDB release 4.1. Search parameters were: fixed carbamidomethyl modification of cysteine; variable oxidation of methionine; peptide tolerance ± 1.5 Da; MS/MS tolerance ± 0.8 Da; +1, +2, +3 peptide charge state; single missed trypsin cleavage.Manual validation of Mascot resultsAdditional manual validation of the proteins identified by Mascot was carried out on the 1-DE and 2-DE results. Proteins identifications that were based on a single peptide and proteins that returned a Mascot score < 60 were accepted if: a matching peptide possessed an individual ion score above the significant threshold for identity or extensive homology (typically > 44); or upon manual inspection of individual peptide MS/MS spectra at least 60% of the candidate y-ions were at a minimum signal to noise ratio of 10%. Spectra that failed to pass either rule were regarded as false positive identifications, which can result from an accumulation of several peptides with low ion scores.Sample preparation for MudPITA pellet of 109 tachyzoites resuspended to approximately 800 μg/ml in 500 μl 100 mM Tris buffer pH 8.5 were lysed by three cycles of freeze/thaw and the Tris-soluble and insoluble protein fractions separated at 16,000 g for 30 minutes. Digestion of soluble fractions: MS compatible detergent Invitrosol was added to 1% (v/v), the solution heated to 60°C for 5 minutes, vortexed for 2 minutes, denatured with 2 M urea, reduced with 5 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), carboxyamidomethylated with 10 mM iodoacetamide, followed by addition of 1 mM CaCl2 and trypsin at a ratio of 1:100 (enzyme:protein) and incubated at 37°C overnight. Digestion of insoluble fractions: 10% (v/v) Invitrosol was added to the pellet, which was heated to 60°C for 5 minutes, vortexed for 2 minutes and sonicated for 1 h. The sample was diluted to 1% (v/v) Invitrosol with 8 M urea/100 mM Tris/HCl pH 8.5, reduced and carboxyamidomethylated as before, and digested with endoproteinase Lys-C for 6 h. The solution was diluted to 4 M urea with 100 mM Tris/HCl pH 8.5 and digested with trypsin as described above.Mass spectrometric analysis by MudPITFive soluble replicates and four insoluble samples were each subjected to MudPIT analysis with modifications to the method of Link et al. [44], using a quaternary Agilent 1100 series HPLC coupled to a Finnigan LTQ-ion trap mass spectrometer (Thermo, San Jose, CA, USA) with a nano-LC electrospray ionization source [45]. Peptide mixtures were resolved by strong cation exchange LC upstream of reverse phase LC as described [46]. Each sample (approximately 100 μg) was loaded onto separate microcolumns and resolved by fully automated 12 step chromatography. Protein databases: a Toxoplasma database was assembled (see above). To identify contaminant host proteins, the parasite database was supplemented with a contaminant database (the complete prokaryote and mammalian databases from NCBI). To estimate the amount of false positives, a reverse database was added [47]. Poor quality spectra were removed from the dataset using an automated spectral quality assessment algorithm [48]. Tandem mass spectra remaining after filtering were searched with the SEQUEST algorithm version 27 [49]. All searches were in parallel and were performed on a Beowulf computer cluster consisting of 100 1.2 GHz Athlon CPUs [50]. No enzyme specificity was considered for any search. SEQUEST results were assembled and filtered using the DTASelect (version 2.0) program [51], which uses a quadratic discriminate analysis to dynamically set XCorr and DeltaCN thresholds for the entire dataset to achieve a user-specified false positive rate (< 5% peptides false positive in this analysis). The false positive rates are estimated by the program from the number and quality of spectral matches to the decoy database.Bioinformatics predictionPrediction programs used were: SignalP to predict proteins that contain signal peptides; TMHMM to predict transmembrane domains; results returned from PATS, PlasMit, and WoLF PSORT together with release4 gene description and GO cellular component prediction provided by ToxoDB were combined to obtain subcellular localization prediction of proteins.Mapping of proteome data to the genome scaffoldPeptides that hit release4 gene annotation could be directly mounted upon the ToxoDB genome scaffold. Where the database search identified preferentially an alternative gene model or an ORF, the sequences were mapped onto the genome using the following algorithm: rule 1, if all the peptides from the alternative models could be mapped to a release4 gene, the release4 annotation is adopted and this is termed a 100% match; rule 2, if more than 50% of the peptides from an alternative model can be mapped to an official release4 gene, this is considered a valid mapping and the matching peptides are aligned with the corresponding release4 gene; rule 3, if a certain set of peptides from an alternative model can be mapped to more than one release4 gene, the gene that can host most peptides will be reported; rule 4, alternative models not conforming to rule 2 will then be mapped to ORFs; rule 5, an alternative model will be mapped to an ORF only if 100% of the peptides can be mapped to that ORF. If 100% of the peptides from the alternative model cannot be mapped to a single release4 gene (rule 1) or to a single ORF (rule 5), the peptides are also mapped to the alternative gene model (for example, TgTwinscan, TgGLEAN, and so on), which can be viewed in GBrowse by selecting the relevant option. This enables ToxoDB users to directly visualize proteomics evidence for alternative gene annotation. All raw data associated with this manuscript may now be downloaded from the Tranche Project [52], using the following hash: Ulv/yTYTaaHin5Tv4InpsgoUY1uTJQtdoLRi9HbdtypXqztv+BiVE/wZieBkqu6d3kU20Vyejo0HYCfswgwiGyPHQPAAAAAAAAOhng==Abbreviations1-DE, 1 dimensional electrophoresis; 2-DE, two-dimensional electrophoresis; ASB-14, amidosulphobetaine-14; DTT, dithiothreitol; EST, expressed sequence tags; GO, Gene Ontology; LC, liquid chromatography; LC-MS/MS, liquid chromatography linked tandem mass spectrometry; MIPS, Munich Information Centre for Protein Identification; MS/MS, tandem mass spectrometry; MudPIT, multidimensional protein identification technology; ORF, open reading frame.Authors' contributionsJMW and SJS conceived and designed the experiments. DX and HP performed the experiments. JY, BB, ARJ and DSR provided analysis tools and software. DX, SJS and ARJ analyzed the data. SJS, ARJ, DX and JMW wrote the paper.Additional data filesThe following additional data are available with the online version of this paper. Data files 1 and 2 are 2-DE gel images showing the spot numbering system that accompanies Figures 1 and 2. Additional data files 3 and 4 are tables listing the MS data and protein identifications corresponding to Figures 1 and 2. Additional data files 5 and 8 are tables listing the MS data and protein identifications (redundant and non-redundant, respectively) for the 1-DE separation illustrated in Figure 3. Additional data file 6 is a 1-DE gel image of Tris-fractionated proteins, and Additional data files 7 and 9 are tables listing the corresponding MS data and protein identifications (redundant and non-redundant, respectively). Additional data files 10 and 11 are tables listing the MS data and redundant protein identifications for soluble and insoluble phase proteins analyzed by MudPIT. Additional data files 12 and 13 are tables listing the protein identifiers corresponding to Figure 5a, b. Additional data file 14 is a pie chart illustrating functional categories for alternative gene models and ORFs.Supplementary MaterialAdditional data file 1Soluble proteins from 2.53 × 108 tachyzoites (516 μg protein) resolved by IEF over a narrow linear pH 3-10 range followed by molecular mass on a 12.5% (w/v) acrylamide gel under denaturing conditions. Protein spots are visualized using colloidal Coomassie. Individual spots are numbered and the corresponding mass spectrometric data are detailed in Additional data file 3.Click here for fileAdditional data file 2Soluble proteins from 1 × 108 tachyzoites (200 μg protein) resolved by IEF over a narrow linear pH 4-7 range followed by molecular mass on a 12.5% (w/v) acrylamide gel under denaturing conditions. Protein spots are visualized using colloidal Coomassie. Individual spots are numbered and the corresponding mass spectrometric data are detailed in Additional data file 4.Click here for fileAdditional data file 3The spot number, matching gene annotation and description, Mascot score, sequence coverage and number of matching peptides are given. Further information concerning peptide sequences is available at ToxoDB. For consistency, where the release4 annotation is not identified by the peptide evidence, TwinScan gene annotation is given in preference to other alternative gene annotations assuming the returning Mascot score is equivalent (and in Additional data files 4, 5, 7, 8 and 9).Click here for fileAdditional data file 4The spot number, matching gene annotation and description, Mascot score, sequence coverage and number of matching peptides are given. Further information concerning peptide sequences is available at ToxoDB. For consistency, where the release4 annotation is not identified by the peptide evidence, TwinScan gene annotation is given in preference to other alternative gene annotations assuming the returning Mascot score is equivalent.Click here for fileAdditional data file 5Listed in the columns (from left to right) are: the gel slice number, ranking of each protein hit returned from the Mascot search for that gel slice, corresponding gene annotations and descriptions, Mascot scores, number of matching peptides to each protein and sequence coverage. Further information concerning peptide sequences is available at ToxoDB. For consistency, where the release4 annotation is not identified by the peptide evidence, TwinScan gene annotation is given in preference to other alternative gene annotations assuming the returning Mascot score is equivalent.Click here for fileAdditional data file 6SDS-soluble proteins from 9.85 × 107 tachyzoites previously fractionated into Tris-soluble (120 μg) and Tris-insoluble (130 μg) fractions were resolved on a 12% (w/v) acrylamide gel under denaturing conditions as follows: protein standards (lane 1), Tris-insoluble protein (lane 2) and Tris-soluble protein (lane 4). Proteins were visualized using colloidal Coomassie. The masses of the protein standards and the position of every gel slice are shown.Click here for fileAdditional data file 7Listed in the columns (from left to right) are: the gel slice number, ranking of each protein hit returned from the Mascot search for that gel slice, corresponding gene annotations and descriptions, Mascot scores, number of matching peptides to each protein and sequence coverage. Further information concerning peptide sequences is available at ToxoDB. For consistency, where the release4 annotation is not identified by the peptide evidence, TwinScan gene annotation is given in preference to other alternative gene annotations assuming the returning Mascot score is equivalent.Click here for fileAdditional data file 8Listed in the columns (from left to right) are: the gene annotations and descriptions of each protein, the highest individual Mascot score, sequence coverage and number of matching peptides returned for that protein from all the gel slices in which it appeared, and the gel slice number that this refers to. Individual peptide amino acid sequence, MS scores and a measure of the total sequence coverage obtained is available at ToxoDB. For consistency, where the release4 annotation is not identified by the peptide evidence, TwinScan gene annotation is given in preference to other alternative gene annotations assuming the returning Mascot score is equivalent.Click here for fileAdditional data file 9Listed in the columns (from left to right) are: the gene annotations and descriptions of each protein, the highest individual Mascot score, sequence coverage and number of matching peptides returned for that protein from all the gel slices in which it appeared, and the gel slice number that this refers to. Individual peptide amino acid sequence, MS scores and a measure of the total sequence coverage obtained is available at ToxoDB. For consistency, where the release4 annotation is not identified by the peptide evidence, TwinScan gene annotation is given in preference to other alternative gene annotations assuming the returning Mascot score is equivalent.Click here for fileAdditional data file 10The unprocessed results from MudPIT analysis lists: gene annotations and descriptions for each protein; alternative gene annotation for that region of the scaffold; total Xcorr scores for each protein hit; individual Xcorr scores theoretical mass and pI values and sequences for each individual matching peptide.Click here for fileAdditional data file 11The unprocessed results from MudPIT analysis lists: gene annotations and descriptions for each protein; alternative gene annotation for that region of the scaffold; total Xcorr scores for each protein hit; individual Xcorr scores theoretical mass and pI values and sequences for each individual matching peptide.Click here for fileAdditional data file 12List of protein identifiers according to subcellular localization category. Number of non-redundant proteins is shown in brackets.Click here for fileAdditional data file 13List of protein identifiers according to functional category. Number of non-redundant proteins is shown in brackets.Click here for fileAdditional data file 14The amino acid sequences of alternative genes and ORFs were submitted to BlastP and results returning e-values < e-30 were considered. Homology to apicomplexan proteins was prioritized when deciding the protein description to be used to assist the assignment of functional category. Sequences returning no significant BlastP result or with a description 'hypothetical protein' were searched against Amigo Blast [54] to determine the potential GO classification. The same e-value cut-off was applied. The above information was then used in conjunction with InterPro and independent literature searches to assign a MIPS category within the FunCatDB functional catalogue. Note: protein fate includes protein folding, modification and destination.Click here for file\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2531087.json

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+{
+    "id": "PMC2531087",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531087\nAUTHORS: Bruce Crawford, Didier Bouhassira, Audrey Wong, Ellen Dukes\n\nABSTRACT:\nBackgroundNeuropathic pain results from a nerve lesion or nerve damage. Because it is a subjective experience, patient-reported outcomes may measure both the symptoms and impact on the patient's life. The purpose of this study was to determine whether the Neuropathic Pain Symptom Inventory (NPSI) adequately assesses neuropathic pain symptoms in patients with diabetic peripheral neuropathy, post-herpetic neuralgia, trigeminal neuralgia, and sciatica across multiple cultures.MethodsFrom data collected from 132 subjects in 6 countries, qualitative research methods identified their most important symptoms (and verbal descriptions) associated with neuropathic pain. A core set of commonly described symptoms spanning multiple cultures was also described. Moderators using a semi-structured discussion guide conducted focus groups consisting of patients in the U.S., Brazil, Japan, China, Finland, and Spain to elicit concepts that were most important and relevant (concept elicitation phase). Study subjects ranked the importance of each neuropathic pain symptom, completed the NPSI, and commented on its ability to capture key symptoms (face and content validation phase).ResultsDescriptive terms for sensations of neuropathic pain were similar in all countries; burning, electric shocks, and pins and needles were among the most-common sensations. Individuals with neuropathic pain experienced all sensations that were included in the NPSI. They also tended to describe pins and needles and numbness interchangeably, perhaps reflecting the relative number of DPN subjects on study.ConclusionBased on data from these focus groups, the NPSI is an acceptable instrument for assessing neuropathic pain.\n\nBODY:\nBackgroundNeuropathic pain results from a nerve lesion or nerve damage and may be experienced as burning, electric shock-like, sharp stabbing pains that come and go, deep aches that make sleep or normal activities difficult, or very sensitive skin that reacts to even a slight touch [1,2]. These sensations not only affect the sensory system, but also translate into a wider impact on patients' health related quality of life in terms of alterations in sleep patterns, concentration and mood. Neuropathic pain has been defined by the International Association for the Study of Pain as pain initiated or caused by a primary lesion or dysfunction of the nervous system [3]. Due to the fact that some researchers find this definition overly broad, neuropathic pain has also been characterized as pain caused by lesions of the peripheral or central nervous system (or both) that manifest sensory symptoms or signs [4]. The assessment of neuropathic pain is often complex, given that it is associated with a wide variety of chronic diseases or conditions such as diabetes, carpal or ulnar nerve entrapments, sciatica, spinal cord injury and neuralgia [5].Neuropathic pain is a subjective experience and the use of patient-reported outcomes (PROs) in measuring symptoms and their manifestation into the patient's life is important. There are several evaluative instruments dealing with neuropathic pain [6-9]. Selecting appropriate measures for the complex assessment of neuropathic pain is challenging. Regulatory agencies have developed guidelines that direct researchers on the development and validation of PRO measures [10,11]. In order for an instrument to be considered well developed, the new guidelines have specified several key points. The development of the instrument must include patient involvement to assist in developing the concepts to be measured or, as the guidelines infer, the question generation process would be incomplete. A wide range of patients should be included in the development of a questionnaire to ensure a representative sample and variations in population characteristics. Following the development of the questions, it is important to review these questions with patients to ensure their clarity and relevance. A questionnaire is not considered valid until the statistical properties have been tested.The new guidelines direct researchers on the validation steps to ensure the measurement properties are adequate for use in clinical trials. Regulatory agencies want to be sure the questionnaire reliably measures the concepts it was designed to measure. It should be noted, however, that the statistical testing of the questionnaire should guide the development and not dictate which items remain in the questionnaire. Relevance to the patient and clinical importance should always be considered. Most questionnaires were developed solely based on clinical expert opinions regarding which symptoms subjects experience and not the patients' perspective on treatment outcomes – an important scientific standard in questionnaire development [10]. In addition, perceptions and descriptions of neuropathic pain might possibly differ between cultures. Thus, to ensure that the questionnaire is suitable for use in worldwide clinical trials, it should not reflect cultural bias.This study evaluates the face and content validity of the Neuropathic Pain Symptom Inventory (NPSI) [8]. The NPSI was developed to assess more specifically the different components of neuropathic pain syndromes (i.e. spontaneous ongoing and paroxysmal pain, evoked pain, paresthesia/dysesthesia). This self-questionnaire includes ten items related to different pain descriptors (e.g. burning, squeezing, electric-shock, stabbing, tingling) allowing the assessment of the different dimensions of neuropathic pain and two items on frequency and duration of pain. Each of the items has a recall of the past 24 hours and items are rated on an 11-point numeric rating scale anchored by 0: No (symptom) and 10: Worst (symptom) imaginable. We employed qualitative research methods to identify symptoms deemed most important to the subjects affected by neuropathic pain and the manner in which the subjects describe those symptoms. Because the NPSI may be used to study several forms of neuropathic pain, it is important to establish a core set of neuropathic pain symptoms. Therefore, this assessment focuses on a core set of symptoms commonly described as symptoms in neuropathic pain that also span multiple cultures.The objective of this study was to determine if the NPSI adequately assesses neuropathic pain symptoms, and is acceptable and relevant to patients with diabetic peripheral neuropathy (DPN), post-herpetic neuralgia (PHN), trigeminal neuralgia (TN), and sciatica across multiple, diverse cultural norms.MethodsRecruitmentFocus groups in six countries (U.S. [English], Brazil [Portuguese], Japan [Japanese], China [Mandarin], Finland [Finnish], Spain [Spanish]) were designed to elicit concepts that were most important and relevant to patients with neuropathic pain. Subjects were recruited through pain specialists via recruitment agencies. The recruitment agencies initiated contact with pain specialists who invited subjects to participate in the study. Subjects received an informational letter outlining the purpose of the study and the extent of their involvement, and physicians obtained informed consent prior to study. Both subjects and their physicians were required to complete a case report form (CRF) that included clinician and subject contact information and ensured the eligibility of the subject through a list of inclusion and exclusion criteria. Subjects were informed that the focus group session would last approximately two hours. The CRFs were reviewed for completeness and patient eligibility prior to beginning the focus group sessions.Six to ten subjects were recruited for each focus group. An attempt was made to recruit subjects of differing age, gender, and ethnicity (the latter only in the U.S.). Subjects with mild to severe neuropathic pain were included to capture the full spectrum of patient pain.Inclusion/exclusion criteriaStudy inclusion criteria included: 18 years of age or older; diagnosed with DPN, PHN, TN, or sciatica; able to discern his/her neuropathic pain from any concomitant pain (e.g., joint pain) as determined by their physician; and ability to participate in a two-hour focus group discussion. In addition, subjects met at least three of the following inclusion criteria (abstracted from the ID Pain [12]) to verify the presence of neuropathic pain: described his/her pain as feeling like pins and needles; described his/her pain as feeling like hot/burning; described his/her pain as feeling numb; has described his/her pain as feeling like electrical shocks; and/or reported that his/her pain is worsened by the touch of clothing or bed sheets. Exclusion criteria included: serious mental health or cognition condition(s), including cognitive impairment, severe mental retardation, schizophrenia, and/or physician-assessed clinical depression.Prior to the initiation of the focus groups, subjects completed forms for informed consent and background demographics, as well as pre-focus group questionnaires. These questionnaires asked subjects to list five terms that describe their nerve pain in conjunction with the five most-bothersome symptoms (i.e., \"People feel pain in many ways and people might describe pain using many different terms. We are interested in how you would describe your nerve pain. Please list below five words that you would use to describe your nerve pain;\" and \"Please list below the three most bothersome sensations you feel related to your nerve pain.\"). Collecting this information spontaneously prior to discussing the topic with other subjects via questionnaire avoids the potential introduction of error through \"yeah-saying\" in the focus groups.Concept elicitation and content validationTrained moderators conducted the focus group sessions using a semi-structured discussion guide. Prior to the start of the focus group, the moderators explained the purpose of the study, reassured the subjects of the confidentiality of their responses, encouraged the subjects to take their time with their responses, and allowed all subjects an opportunity to share their views with the group. The moderator informed the participants that the focus group sessions would be audio- and/or video-recorded as stated in the consent form that each participant had signed prior to the focus group. The focus group guide consisted of: 1) a concept elicitation phase, and 2) face and content validation phase. During the concept elicitation phase, subjects received open-ended questions about their neuropathic pain experiences, focusing on symptoms they experienced due to their neuropathic pain. Subjects identified and described such sensations in detail. Initially, subjects responded spontaneously to these questions. If sensations previously described in the questionnaire were not mentioned spontaneously, the moderator probed the subjects to determine the accurateness of the sensations. These questions were asked prior to the content validation phase of the interview to ensure that the subjects were not unduly biased by the sensations covered in the NPSI. This allowed for a pure assessment of symptoms prior to the face and content validation of the questionnaire and a more guided assessment of symptoms during the second phase of the focus group.During the concept elicitation phase, the importance of each neuropathic pain symptom was ranked by patients detailing the \"most bothersome\" sensation they experience. During the face and content validation phase of the focus groups, the subjects completed the NPSI and commented on the extent to which the questionnaire captured key symptoms associated with neuropathic pain. The purpose of this phase of the focus groups was to ensure: 1) the relevance of the concepts covered by the questionnaire, 2) the questionnaire's comprehensiveness and ease of understanding, and 3) the applicability/acceptability of the items.Transcription/translationTranscriptions were produced from the audiotapes of the sessions, and verbatim subject comments were analyzed. Recordings in Japanese, Spanish, Portuguese, and Chinese were transcribed into the respective native language prior to English translation. The English transcripts of the other countries' focus group data were then analyzed. The Finnish tapes were transcribed into Finnish and then analyzed in the native language. Subject quotes were grouped together by symptom and compared to the symptoms included in the NPSI.Coding schemes were developed to translate descriptions of patient characteristics into thematic trends for data analysis. The thematic coding scheme underwent iterations as the research team coded the preliminary data. Initial coded material was aggregated into broader core categories and analyzed using grounded theory methods [13]. For the concept elicitation sections of the focus groups, each subject comment was assigned a \"classification\" and \"domain\" and incorporated into a domain mapping grid. The classifications and domains identified, along with examples of subject quotes, were used as a basis for determining whether all relevant symptoms were included in the NPSI.Results and discussionOne hundred and thirty-two subjects from six countries were interviewed (Table 1), Background demographics, including age and gender are summarized in Table 2. The type of neuropathic pain and clinician-rated severity of pain are included in Table 3.Table 1Focus Group PopulationsCountryNumber of focus groupsTotal number of subjectsUnited States650Brazil1 (plus 10 individual in-depth interviews)a16China218Finland217Spain216Japan213a Conducted in place of a focus group due to scheduling conflicts.Table 2Focus Group DemographicsDemographic InformationU.S. (N = 50)Brazil (N = 16)China (N = 18)Finland (N = 17)Spain (N = 16)Japan (N = 13)n (%)n (%)n (%)n (%)n (%)n (%)Gender- Female24 (48)9 (56)8 (44)11 (65)13 (81)5 (38)- Male26 (52)6 (38)9 (50)6 (35)3 (19)8 (62)- Missing Data0 (0)1 (6)1 (6)0 (0)0 (0)0 (0)Age- Range19–81 years50–76 years28–61 years43–90 years23–78 years54–80 years- Mean52 years62 years47 years70 years66 years66 years- Median51 years61 years66 years61 years72 years64 yearsEducation*- Less than high school4 (8)9 (56)9 (50)15 (88)10 (63)3 (23)- High school diploma/Some college28 (56)7 (44)3 (17)--4 (25)5 (38)- College or university degree (2 or 4 year)16 (32)--6 (33)--2 (12)4 (31)Marital Status**- Married31 (62)10 (63)18 (100)10 (59)9 (56)12 (92)- Not married19 (38)6 (37)--7 (41)7 (44)--* Note: Two patients from the U.S. did not respond; two patients from Finland did not respond; one patient from Japan did not respond.**Note: One patient from Japan did not respond.Table 3Focus Group Health InformationHealth InformationU.S. (N = 50)Brazil (N = 16)China (N = 18)Finland (N = 17)Spain (N = 16)Japan (N = 13)n (%)n (%)n (%)n (%)n (%)n (%)Type of Neuropathic Pain- Diabetic Peripheral Neuropathy18 (36)14 (88)5 (28)0 (0)1 (6)5 (38)- Post-Herpetic Neuralgia10 (20)1 (6)6 (33)5 (29)10 (63)8 (62)- Trigeminal Neuralgia8 (16)1 (6)7 (39)12 (71)4 (25)0 (0)- Sciatica14 (28)0 (0)0 (0)0 (0)0 (0)0 (0)- Missing data0 (0)0 (0)0 (0)0 (0)1 (6)0 (0)Clinician-rated pain level- Mild15 (30)3 (19)1 (6)0 (0)0 (0)3 (23)- Moderate26 (52)12 (75)13 (72)1 (6)9 (56)6 (46)- Severe9 (18)1 (6)4 (22)16 (94)7 (44)4 (31)In the U.S., the majority of the subjects (72%) were Caucasian. The remaining participants were African American (8%), Hispanic/Latino (11%), and from other ethnic groups (3%). As illustrated in Table 2, there was some variability by country in both educational level and marital status. Ethnicity was not collected in the other countries due to the ethnic homogeneity for each country. It should be noted that with the exception of the U.S., focus groups were conducted in or around major cities – Sao Paulo, Beijing and Shanghai, Seinajoki (smaller city in western Finland), Madrid and Tokyo.Pre-focus group findingsTable 4 summarizes the spontaneous, independent report of symptoms by subjects on the pre-focus group questionnaire, as described in Methods. The most frequently listed words to describe neuropathic pain were \"burning,\" \"electric shock,\" \"numbness,\" and \"tingling\"; however, not all of the subjects listed sensations.Table 4Sensations of Neuropathic Pain Included in the NPSI Compared to Sensations Reported on the Pre-Focus Group QuestionnaireNeuropathic PainSensations of  Included in the NPSISensations of Neuropathic Pain Reported on Pre-Focus Group QuestionnaireU.S. (n = 50)Brazil (n = 16)China (n = 18)Finland (n = 17)Spain (n = 16)Japan (n = 13)Burning1257151Squeezing------3----Pressure1--1312Electric Shocks535745Stabbing5--7--51Pins and Needles5--15--6Tingling65112----Non-NPSI SensationsNumbness927223Prickling2--------2Itchiness7------84Sharp9----------Shooting5----3----Throbbing2----------Stinging41--------Piercing--1--------Cramps--1----1--Cutting----35----Hot----11----Pulsating------1----Drilling------1----\"Squeezing\" and \"pressure\" were the least likely sensations on the NPSI to be elicited spontaneously on the pre-focus group questionnaire. \"Pressure\" was reported in every country except Brazil and \"squeezing\" was only mentioned in Finland.All sensations covered in the NPSI were mentioned spontaneously as being most bothersome on the pre-focus group questionnaire except for squeezing. The most frequent notations of bothersome were burning, tingling, and electric shocks.Focus group findingsPhase 1During the focus groups, the most common spontaneous descriptions were burning, electric shocks, numbness, and pins and needles. Subjects often used terms interchangeably; for example, in the U.S., \"tingling\" and \"numbness\" were described as \"pins and needles.\"In Brazil, all symptoms on the NPSI were spontaneously mentioned in the focus group except \"squeezing\" and \"tingling.\" After probing, subjects also reported experiencing \"squeezing.\" \"Tingling\" was the only sensation not mentioned by the subjects in Brazil. \"Cramps\" were described as \"similar to twinging\" and \"coming after the burning pain.\" After a discussion with a professional translator, it was discovered that \"twinging\" might be the English translation of the Brazilian word for \"tingling.\" One patient described \"twinging\" as \"stabbing by needles.\"In China, subjects also used the terms \"heart stabbing,\" \"needle through heart,\" \"tremble,\" and \"bursting\" to describe their pain. Interviewers in China noted that these terms should not be interpreted literally. \"Bursting\" implies a \"sudden, strong, and unbearable\" feeling of pain. The two terms referring to the heart do not mean that the heart is in pain. When speaking about pain, the Chinese are more likely to relate extreme pain with the heart because they believe the heart is the most critical and sensitive part of the body.In Spain, the two sensations of \"pins and needles\" and \"stabbing\" were combined into one term as \"stabbing pins on fire\" (n = 8). One subject defined it as if \"hundreds, thousands of pins on fire (are) stuck into my body.\"Table 5 summarizes the pain sensations experienced by the focus group members that they spontaneously described. The symptoms of the NPSI were consistently reported within the focus groups with the exception of \"squeezing.\" Although \"squeezing\" was reported in the U.S., Finland and Japan, few subjects stated this as a spontaneous expression of their pain. \"Squeezing\" was only spontaneously mentioned by one subject and four subjects mentioned \"squeezing\" while describing other neuropathic pain sensations.Table 5Sensations Reported in the Focus GroupsSensations ofNeuropathic Pain  Included in the NPSISensations of Neuropathic Pain Spontaneously Mentioned by Subjects During the Focus GroupsU.S. (n = 50)Brazil (n = 16)China (n = 18)Finland (n = 17)Spain (n = 16)Japan (n = 13)Burning14102211--Squeezing1----5--1Pressure63--552Electric Shocks103210156Stabbing62--183Pins and Needles876154Tingling622312All of the sensations of neuropathic pain included in the NPSI (e.g., burning, squeezing, pressure, electric shocks, stabbing, pins and needles, and tingling) were spontaneously mentioned by subjects during the focus groups. Of the sensations included in the NPSI, \"burning,\" \"pins and needles,\" and \"electric shocks\" were most frequently mentioned by subjects in the focus groups. Subjects in China did not spontaneously mention three of the seven items (e.g., squeezing, pressure, and stabbing).In addition to symptoms included on the NPSI, subjects also frequently mentioned \"numbness\" and \"sharp\" as sensations they experienced, although \"sharp\" was only mentioned in the U.S.Patients in each country consistently described their pain with a single statement. Subjects in the U.S. used \"burning,\" \"electric shocks,\" and \"sharp\" while those in Spain used \"electric shocks\" or \"sharp\" only. Finnish and Japanese subjects also described their pain as \"electric shocks,\" In addition, Japanese subjects used the term, \"pins and needles.\"The two most bothersome sensations in the U.S. were burning and electric shocks while the two most bothersome sensations in Brazil were cramps and pins and needles. The most bothersome sensations for Spanish subjects were either electric shocks or \"stabbing pins on fire.\" Interestingly, subjects in China defined their worst pain by the emotions they felt or their inability to sleep in addition to the type and duration of the pain episode.Review of the Neuropathic Pain Symptom Inventory questionnaireThe majority of the subjects did not raise any concerns with the NPSI: only three subjects mentioned that the recall period was too short, one subject felt that the questionnaire was confusing and another thought it did not capture all of their symptoms. Subjects responded positively when asked if the questionnaire was easy to understand, though one person reported that they did not know what was meant by \"squeezing\" pain. Next, subjects were asked which words (or kanji characters) they thought were above a sixth-grade reading level. The majority of subjects in other countries stated no concerns; however, words that some U.S. subjects thought were above a six-grade reading level included imaginable, neuropathic, provoke, severity, spontaneous, stimulation, and sensation. Although not thought to be above a six-grade reading level, Japanese subjects suggested that \"pressure\" was a concept that may be difficult to understand. However, no changes to the NPSI were consistently suggested by focus group subjects.In China, four subjects felt that the questionnaire did not adequately reflect Chinese and/or Asian culture, and they suggested using a simplified NPSI. Because pain is not judged on a numerical scale, patients did not define their pain in such detail. Instead, subjects in China typically used descriptive terms (\"mild,\" \"moderate,\" or \"severe\") rather than numbers to quantify pain. However, five individuals felt that the NPSI was an acceptable tool, even if it incorporated a scale to measure pain.ConclusionThe focus groups and interviews consisted of 2 phases: 1) concept elicitation, and 2) face and content validation. The information gathered from the focus groups in other countries (e.g., Japan, Brazil, China, Finland, and Spain) was consistent with that from group in the U.S. Descriptive terms for sensations of neuropathic pain were similar in all countries studied. Burning, electric shocks, and pins and needles were among the most-common sensations. Based on feedback from focus group subjects during the concept elicitation phase, all sensations included in the NPSI are indeed experienced by people with neuropathic pain. During the focus groups or individual interviews, subjects used the terms burning, electric shocks, and pins and needles.Numbness was also consistently mentioned. Although \"numbness\" is not a true pain descriptor but is related to non-painful paresthesia/dysesthesia, the occurrence of numbness as a frequently reported sensation reflects the number of DPN subjects in the focus groups, as this sensation is typically experienced in DPN. Subjects also used the words numbness and pins and needles interchangeably to describing pain symptoms. Because pins and needles are already included in the NPSI, adding numbness should be considered when a DPN-specific questionnaire is required. Because numbness would not be a component of the validated scoring algorithm, this issue would be considered separately. Similarly, \"itchiness\" is not a true pain descriptor. In the validation of the NPSI [8], \"itchiness\" was found to be an unreliable item and therefore was removed. This study, unfortunately, was not designed to evaluate the \"global\" reliability of responses and therefore, we cannot recommend its inclusion at this time. The descriptor of \"squeezing\" was not consistently reported across cultures; however, \"pressure\" was reported more consistently. These two descriptors have been found to belong to the same pain dimension [8] – spontaneous ongoing pain, with similar factor loadings (0.88 and 0.87, respectively). It is therefore thought that these two descriptors will complementarily assess the spontaneous ongoing pain symptoms.This study was not able to evaluate the differing etiology of pain in the analysis due to the separation of the participant's personal health information from the focus group transcripts. It is likely that subjects across different etiologies describe their pain slightly differently. It would have also been interesting to investigate the terminology utilized by subjects across cultures with the same etiology. As the objective of this study was to evaluate the adequacy of the NPSI for use in different neuropathic pain etiologies in different countries, the results support the broad objective. This is the first study to the knowledge of the authors to confirm such a \"universality\" of core neuropathic pain descriptors across etiologies and cultures. This study suggests that the small impact of culture on neuropathic pain expression may be related to its specific pathophysiologic mechanism; confirming the notion that neuropathic pain is a specific category of chronic pain that deserves special attention.In conclusion, the information collected during the focus groups and their analyses demonstrate that the NPSI is an acceptable instrument for assessing neuropathic pain worldwide. Country-specific terms might further enhance its applicability.AbbreviationsCRF: Case Report Form; DPN: Diabetic Peripheral Neuropathy; NPSI: Neuropathic Pain Symptom Inventory; PHN: Post-herpetic Neuralgia; PROs: Patient-reported Outcomes; TN: Trigeminal Neuralgia.Competing interestsBC and AW are employees of Mapi Values, an outcomes research consulting firm. ED is an employee of Pfizer Inc. DB has received funding for research and speaking engagements from numerous pharmaceutical companies. There are no other competing interests.Authors' contributionsBC and ED were responsible for the design and execution of this study. AW was the primary analyst. DB assisted in the interpretation of the results. All co-authors assisted in drafting the manuscript.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2531094.json

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+{
+    "id": "PMC2531094",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531094\nAUTHORS: Vinay Singh, Pankaj Singh, Amit Sharma, Jay Sarkar\n\nABSTRACT:\nAcute constriction ring syndrome (ACRS) is a rare clinical condition characterized by formation of a circumferential constriction ring around an appendage or genitalia. Cases are mostly reported in infants and young children. Early recognition and a definitive treatment are of paramount importance in order to avoid irreversible ischemia and possible auto-amputation. We describe a case of a 14-month-old child presented to casualty with a history of refusal to feed and inconsolable cry. Parents noticed a recent swelling of left third toe. On careful examination the child was found to have an acquired constriction ring secondary to a tightly wrapped hair around left third toe. An urgent surgical decompression was done by the orthopaedic team with complete resolution of symptoms. We summarized the pathophysiology of ACRS underlining the need of awareness in treating physicians. The possible medico legal implications should be kept in mind bearing a suggested link with non-accidental injury.\n\nBODY:\nBackgroundAcquired constriction ring syndrome (ACRS) is more commonly known as hair tourniquet syndrome or hair thread tourniquet syndrome [1]. It is clinical condition characterized by circumferential constriction of an appendage or genitalia by human hair, synthetic fibre or a thread [2]. It usually affects infants and has infrequently involved adolescent and cognitively impaired adults [3,4]. Congenital, [1,5] accidental as well as non-accidental [6-8] aetiologies have been proposed by different authors. Lesser toes remain the most common site affected1 followed by fingers and genitalia [8-11].Case reportA 14-months-old baby girl was brought to casualty by mother with an inconsolable cry and refusal to feed in our institute just after Christmas in 2007. Mother noticed a swelling and redness of left third toe while changing her socks in the morning. There was no history of trauma or any congenital deformity. On examination child was afebrile and systemically well. The digit was tender on touch with profound redness and swelling involving the distal phalange. The initial naked-eye examination done in emergency department failed to reveal a definitive cause. The child was referred to orthopaedic team for further opinion. The extensive swelling in an already distressed child was restricting the extent of assessment. Appropriate analgesics were given after establishing an intravenous access. A lens and loupe examination performed in emergency treatment room under light sedation, revealed the presence of a constriction ring at distal inter-phalangeal joint (Fig 1, 2). The skin was breached on dorsal surface of toe with volar surface showing a deeply buried ring of hair. Delayed capillary refill indicated an imminent danger of gangrene secondary to ACRS. The examination of other digits and genitalia was unremarkable. A non-accidental cause was ruled out with the help of paediatricians.Figure 1The constriction ring of third toe can be seen clearly at the level of distal inter-phalangeal joint with redness and oedema.Figure 2The constriction ring is visible in another image with resultant distal oedema and swelling.Child was urgently taken to theatre to avoid further neurovascular compromise. Constriction ring of hair was removed with a pair of fine scissors under general anaesthetic (Fig 3). Hair was examined for the presence of well formed knots. Constriction ring was also decompressed by a small dorsal slit-incision down to the bone avoiding extensor tendons and the wound was left open. The child was comfortable in the postoperative period with a significant reduction of swelling by the next day. Prophylactic oral antibiotics were given for a week to avoid secondary infection. The kid was discharged from clinic in a week's time after complete resolution of symptoms.Figure 3Intraoperative photograph taken after the release of constriction ring. Note the releasing incision at the dorsal surface.DiscussionHair tourniquet syndrome is characterized by circumferential strangulation of an appendages or genitalia by human hairs or fibres. The condition was first described in 1832 [12] and the term hair tourniquet syndrome was first coined by Quinn in 1971[13]. Since then various authors have used eponyms hair thread tourniquet syndrome and acquired constriction ring syndrome to describe the same entity. The exact incidence of the condition is not known but mostly considered to be rare [2]. Whereas there are reports in paediatric, emergency and orthopaedic subspecialty periodicals, the condition failed to attract the attention of general orthropods [2]. The condition primarily involves infants with over 80% of the cases being reported in a population younger than 2 months [1]. However, there are occasional reports of adolescents and adults with cognitive impairment being involved [3,4]. Hair remains the most common causative agent with a reported incidence of 79% in one study [14]. Hair has unique physical characteristics which make it an ideal tourniquet. It is thin, elastic and expansible when wet while constricts as it dries off without losing its tensile strength [1].Etiology of ACRS remains a matter of debate with more then one mechanisms playing role including accidental, [1,2] non-accidental [6-8] and congenital [1,5]. There are some predisposing conditions described in literature indicating the accidental nature. Baby cloths such as mittens and single piece jumpsuits may cause accumulation of hairs and may pose increase risk of ACRS [1,2]. Postpartum excessive hair loss (telogen-effluvium) has been known to predispose risk of ACRS [14,15]. In spite of the name acquired constriction, a group of authors believe ACRS to be of congenital origin [1,5] and the high incidence (> 80%) of cases in early infantile life has been attributed to its congenital origin. The other group, however strongly advocate ARCS to be non-accidental in nature until proven otherwise [6-8]. Authors suggested that non-accidental injury (NAI) to be considered where there is no reasonable explanation for presence of meticulously wrapped constriction ring or presence of well formed knots [5]. ACRS involving limb are mostly accidental and that involving genitals are thought to be associated with NAI [15]. The absence of well formed knots and an unremarkable paediatric assessment effectively ruled out NAI in our case. Contrary to digital variety the genital cases are usually seen in relatively older children ranging from 4–11 years. This age corresponds to the Sigmund Freud's phallic (or clitoral) stage of psychosexual development when kids start toying with their genitalia out of curiosity. The application of a tourniquet around genitalia may be a possible reflection of this psychosexual tendency. Some of the cultural practices also warrant a mention where an intentional tourniquet applied to ward off evil spirits or treat urinary incontinence and nocturnal emissions led to ACRS [8]. The pathophysiology of ACRS can be compared with compartment syndrome. The constriction ring interferes with the distal venous and lymphatic drainage at first, leading to venous engorgement. This progressive oedema further exacerbates the constriction setting a vicious circle ultimately ushering the ischemia and gangrene if not relieved in time [6].Different measures have been suggested to prevent the ACRS. Counselling of postpartum mother is imperative and crucial. It is important to educate her to perform through regular checks of limbs to ensure that no hairs are entangled around fingers and toes of the baby. Mittens, single piece jumpsuits and clothing covering fingers and toes should be regularly checked for presence of loose hairs and should be washed inside out [14].Early recognition with an urgent decompression remains the mainstay of treatment in an established case. Apart from the affected toe, a thorough examination should include all unaffected toes, fingers and genitals in order to rule out simultaneous involvement else where. Decompression may be carried out with fine scissors or using a depilatory cream in initial stages of ACRS when skin is intact and swelling is minimal [1,2]. In advanced stages when swelling is profuse or skin has breached, then it is advisable to carry out urgent complete decompression in theatre preferably under general anaesthetic [2]. Adequate light and magnification will further aid the search for constricting agent which may be lying buried deeply inside the subcutaneous tissues. The skin may re-epithelialize over the buried hair making the exploration further difficult [2]. Once the constricting agent is completely removed the soft tissue constriction ring itself should be decompressed by making a small vertical bone deep incision on dorsal surface avoiding extensor tendons [2]. It is vital to examine the tourniquet for presence of a well formed knot as it would strongly suggest presence of NAI. Decompression in casualty may be performed in early stages of ACRS but must be avoided if skin has breached or there is profound swelling making adequate visualization of constricting agent impossible. Inadequate visualization in fully established ACRS may lead to incomplete release and adverse clinical consequences.ConclusionParental education is of immense value in reducing the incidence of ACRS. For clinicians, it is crucial to treat ACRS as an appendage threatening emergency and perform urgent complete decompression in theatre. Incomplete decompression may lead to further ischemia and auto-amputation. Inconsolable cry and refusal to feed are two most common but equally no-specific symptoms in infants and young children. Clinicians working in acute paediatric settings should be aware of the entity of acquired constriction ring syndrome as a possible cause, after excluding the common ones. NAI should be kept in mind when dealing with ACRS and through examination for presence of knots at time of decompression is important as it may give precious clue to etiology. ARCS, a potentially reversible cause of neurovascular compromise, should be kept in differential diagnosis of acute swelling in an appendage or genitalia of an obscure etiology.AbbreviationsACRS: Acquired constriction ring syndromeCompeting interestsThe authors declare that they have no competing interests.Authors' contributionsVS: Operating surgeon, collected clinical details including photographs, summarised the case history and prepared first draft. PS:Conducted a literature search, design and formatting of final manuscript, including grammar. Verified the authenticity of scientific content. AS: Helped in conducting the literature search and extracting the papers from library and internet. He also contribute in preparation of electronic images and electronic formatting of manuscript. JS: Treating consultant and performed final editing of the manuscript.ConsentA fully informed written consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2531101.json

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+{
+    "id": "PMC2531101",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531101\nAUTHORS: Chandima P Karunanayake, Helen H McDuffie, James A Dosman, John J Spinelli, Punam Pahwa\n\nABSTRACT:\nBackgroundThe objective was to study the association between Non-Hodgkin's Lymphoma (NHL) and occupational exposures related to long held occupations among males in six provinces of Canada.MethodsA population based case-control study was conducted from 1991 to 1994. Males with newly diagnosed NHL (ICD-10) were stratified by province of residence and age group. A total of 513 incident cases and 1506 population based controls were included in the analysis. Conditional logistic regression was conducted to fit statistical models.ResultsBased on conditional logistic regression modeling, the following factors independently increased the risk of NHL: farmer and machinist as long held occupations; constant exposure to diesel exhaust fumes; constant exposure to ionizing radiation (radium); and personal history of another cancer. Men who had worked for 20 years or more as farmer and machinist were the most likely to develop NHL.ConclusionAn increased risk of developing NHL is associated with the following: long held occupations of faer and machinist; exposure to diesel fumes; and exposure to ionizing radiation (radium). The risk of NHL increased with the duration of employment as a farmer or machinist.\n\nBODY:\nBackgroundNon-Hodgkin's Lymphoma (NHL) is a cancer of the lymphatic system [1,2]. Even though NHL is a relatively rare disease, its incidence rates have been increasing worldwide for both men and women. The incidence rates in Canada, for both males and females were increased by about 50% between 1978 and the late 1990s. After the latter time, incidence rates have stabilized. Mortality rates of NHL have followed a similar pattern [3]. Age-standardized rates have increased faster among males than among females [1-5]. A number of factors, including inherited and acquired immunodeficiency states [6] as well as infectious, physical, and chemical agents have been associated with an increased risk for NHL [6,7].Epidemiological studies have reported positive associations between NHL and certain occupations including those of farmers [8-16], pesticide applicators [12,17-20], drivers [21,22], and managers [23,24]. Several studies have reported no association between development of NHL and the agricultural occupations (farmers, agricultural and forestry workers and pesticide applicators [25-27]). Occupational exposures of a priori interest include pesticides [28-33], dusts (metal, wood, paper [8], etc), paints [8,35], diesel exhaust fumes [21,22,34,35], cleaning fluids [8], cutting oils [36], and solvents [37,38]. In this paper, we examined the association between NHL and (1) selected long term occupations, and (2) occupational exposures based on an individual's occupational history, and (3) duration of employment.MethodsDetails of the study design and methodology have been previously published [39-41]. Briefly, we conducted a six province Canadian population based case-control study of men with an incident first diagnosis of NHL between 1991 to 1994; control subjects were frequency matched by age ± 2 years to be comparable with the age distribution of the entire case group (Soft Tissue Sarcoma (STS), Hodgkin's Disease (HD), NHL, and Multiple Myeloma (MM)) within each province of residence. The study had approximately three matched controls for each NHL case. Deceased subjects were ineligible as either cases or controls. All participating control subjects were used in the statistical analysis of each cancer site. Cases were identified from provincial cancer registries – except in Quebec where hospital records were used – and were coded using ICD-O 2nd edition except Quebec which used ICD-O 1st edition [42]. Malignant morphology codes 9591, 9642, 9670–9764, and 9823 were included. A reference pathologist reviewed the tumour tissue slides for 60% of the NHL cases, and confirmed NHL in all but 2% of cases. Cases not confirmed as NHL were eliminated. Control subjects were identified through provincial health insurance programs except in Ontario (telephone listing) and British Columbia (voter's lists), as generally described [39-41].The study design consisted of two stages: Stage 1 was a self-administered postal questionnaire; and Stage 2 was a detailed pesticide exposure information collected via telephone interview. With permission, we modified a pesticide exposure questionnaire developed by Hoar et al. [43] to create the study questionnaire. The results in this manuscript are based on the Stage 1 postal questionnaire only.The postal questionnaire captured demographic details, personal medical history, lifetime occupational history and specific occupational exposures of interest. Occupational information included a list of all full time jobs held by the respondent for at least one year. For each job held, we collected information on job titles, business organization – whether service or industry – and duration of employment. A list of occupational exposures that have been epidemiologically linked to NHL or to one of the other three types of cancers which we studied simultaneously was grouped into dusts, coal products, printing products, paints, metals, pesticides, radiation and miscellaneous. Additional details of exposure to agricultural chemicals in broad classes i.e. herbicides, fertilizers etc, were obtained. Job titles and each industry's coding were provided by Statistics Canada [44].Statistical analysisData were entered into a custom designed SPSS-data entry program. Results were presented as frequencies for categorical variables; mean, standard deviation (SD) for continuous variables for cases and controls were presented separately. We obtained information about the duration of employment (measured in years) for each individual. The occupations were selected for analysis if the occupant worked in a particular occupation at least for one year and at least 2% of cases for that occupational category. Based on that information, we derived two new variables called ever held occupations and long held occupations. Occupations were defined as ever held occupation if respondents worked at least for one year in that occupation. Occupations were defined as long held occupation if respondents worked for 10 years or more in that occupation. Duration of employment is the total of number of years in each long held occupation. A bivariate analysis was conducted to determine the association between each explanatory variable and the NHL outcome. Based on this model, building procedure explanatory variables with p < 0.20 were selected for the multivariate model. Statistically significant (p = 0.05) variables and important explanatory variables were considered for the final multivariate model adjusting for age and province of residence. Conditional logistic regression was used to compute adjusted odds ratios (OR) and 95% confidence intervals (95% CI).EthicsThe letters of informed consent, questionnaires, and all other correspondence with study participants were approved by the relevant ethics agencies in each province. All of the information that could be used to identify study participants remained within each province of origin under the supervision of the provincial principal investigators.ResultsThis study includes responses from 513 cases with NHL and 1506 control subjects. The mean age ± standard deviation (SD) of cases was 57.7 ± 14.0 years and, of the controls, 54.1 ± 16.0 years. More cases (n = 74, 14.4%) than controls (n = 87, 5.8%) had a personal history of cancer other than NHL (ORadj (95 % CI): 2.56 (1.81, 3.62)). There were no significant differences between NHL cases and controls with respect to their education level and to whether they ever lived or worked on a farm. Results are shown in Table 1.Table 1Characterization of study participants stratified by NHL case- control status: demographics and selected medical historyNHL (N = 513)Controls (N = 1506)ORbadj [P1](95% CI)Demographics Mean age ± SD (years)57.7 ± 14.054.1 ± 16.0 Education Levela  University and Vocational28 (6.6)96 (5.5)1.23 (0.81, 1.88)  University94 (18.5)310 (20.8)1.08 (0.68, 1.70)  Vocational111 (21.9)358 (24.1)1.06 (0.67, 1.70)  Elementary/High school274 (54.0)723 (48.6)1.00 Ever lived/worked on a farm  Yes n (%)235 (45.8)673 (44.7)1.02 (0.82, 1.27)  No n (%)278 (54.2)833 (55.3)1.00Medical History Previous diagnosis of Cancer  Yes n (%)74 (14.4)87 (5.8)2.56 (1.81, 3.62)c  No n (%)439 (85.6)1419 (94.2)1.00a 25 missingb Adjusted for age (5 year groups) and provincec Statistically significant results are bold.Table 2 shows the distribution of ever held occupations and long held occupations during a lifetime stratified by case-control status. None of the ever held occupations were statistically significant. The long held occupations (10 years or more) as farmer and machinist showed a significant risk increase for NHL. The adjusted odds ratios (ORadj) and 95% confidence intervals (95% CI) for a long held occupation during the lifetime as farmer and machinist were 1.54 (1.05, 2.27) and 2.21 (1.02, 4.79) respectively. Using four categories (no exposure, < 10 years, 10–20 years, and > 20 years), further models with years in these industries were used to investigate whether or not there is a dose-response relationship between the long held occupation as a farmer and a machinist and NHL (Table 3). A dose-response relationship between duration of exposure as farmer and incidence of NHL was observed. Those who worked as a farmer for more than 20 years were 1.5 times more likely to be diagnosed with NHL than non-exposed subjects. Similarly, we observed a dose-response relationship between duration of exposure as a machinist and incidence of NHL. Those who worked as a machinist for more than 20 years were 2.3 times more likely to be diagnosed with NHL than non-exposed subjects (Table 3).Table 2Adjusted odds ratio (OR) and 95% confidence interval (95% CI) for different occupations (job titles).Job Title (code#)NHL cases n (%)Controls n (%)OR adj a (95% CI)Ever held OccupationsAccountant (1)30 (5.8)81 (5.4)1.21 (0.77, 1.89)Administrator (2)11 (2.1)52 (3.4)0.58 (0.30, 1.15)Carpenter (12)21 (4.1)55 (3.6)1.06 (0.63, 1.79)Clerk (17)14 (2.7)92 (6.1)0.44 (0.24, 0.79)Constructor (19)14 (2.7)78 (5.2)0.51 (0.28, 0.93)Driver (25)55 (10.7)133 (8.8)1.29 (0.91, 1.82)Electrician (26)16 (3.1)47 (3.1)0.99 (0.54, 1.78)Engineer (27)13 (2.5)68 (4.5)0.54 (0.29, 1.02)Factory worker (29)13 (2.5)46 (3.0)1.14 (0.59, 2.17)Foreman (30)11 (2.1)39 (2.6)0.64 (0.32, 1.28)Farmer (31, 33, 89)86 (16.7)230 (15.3)1.14 (0.85, 1.54)Armed forces (138)28 (5.5)92 (6.1)0.76 (0.48, 1.18)Janitor (41)14 (2.7)40 (2.7)1.07 (0.57, 2.02)Labourer (44)31 (6.0)99 (6.6)0.86 (0.56, 1.33)Lumberman (46)17 (3.3)38 (2.5)1.12 (0.61, 2.03)Machinist (47)22 (4.3)49 (3.2)1.41 (0.83, 2.40)Manager (48)63 (12.3)183 (12.1)0.97 (0.70, 1.33)Mechanic (49)26 (5.1)88 (5.8)0.83 (0.52, 1.31)Salesman (73)44 (8.6)127 (8.4)1.06 (0.73, 1.53)School Teacher (74)31 (6.0)88 (5.8)0.96 (0.62, 1.48)Welder (86)13 (2.5)33 (2.2)1.25 (0.64, 2.44)Office worker (97)17 (3.3)68 (4.5)0.70 (0.40, 1.22)Equipment hander (134)14 (2.7)37 (2.5)1.34 (0.70, 2.56)Long held OccupationsAccountant (1)20 (3.9)41 (2.7)1.39 (0.79, 2.42)Driver (25)27 (5.3)48 (3.2)1.45 (0.88, 2.37)Farmer (31, 33, 89)50 (9.8)106 (7.0)1.54 (1.05, 2.27)cMachinist (47)12 (2.3)16 (1.1)2.21 (1.02, 4.79)cManager (48)31 (6.0)96 (6.4)0.86 (0.56, 1.32)Mechanic (49)15 (2.9)49 (2.2)1.00 (0.99, 1.02)# Statistics Canada. Standard occupational classification. Ottawa: Minister of Supply and Services, 1980.a All odds ratios were adjusted for age and province of residence.c Statistically significant results are bold.Table 3Duration of exposure as a farmer and machinist and risk of NHLDuration (in years)NHL (N = 513)Control (N = 1506)OR (95% CI)an (%)n (%)Job Title: FarmerNo exposure427 (83.2)1276 (84.7)1.00<10 years36 (7.0)124 (8.2)0.84 (0.51, 1.41)10–20 years7 (1.4)23 (1.5)1.40 (0.57, 3.43)> 20 years43 (8.4)83 (5.5)1.55 (1.02, 2.36)cJob Title: MachinistNo exposure491 (95.7)1457 (96.7)1.00<10 years10 (1.9)33 (2.2)0.75 (0.30, 1.88)10–20 years2 (0.4)4 (0.3)1.77 (0.31, 10.22)> 20 years10 (1.9)12 (0.8)2.33 (1.00, 5.52)ca all odds ratios were adjusted for age and province of residence.c Statistically significant results are bold.Of the 45 specific occupational exposures grouped into six classes (dusts, coal products, printing, paints, metals and miscellaneous), only exposure to diesel exhaust fumes showed an association with NHL (Table 4). Ever exposure to solvents and exposure to wood or paper dust were not associated with NHL. Ever exposure to ionizing radiation (radium) showed a significant association with the risk of NHL incidence (OR adj (95% CI): 3.26 (1.38, 7.73)).Table 4Adjusted odds ratio (OR) and 95% confidence interval (95% CI) for different occupational exposures.NHL (N = 513)Control (N = 1506)Exposurenb%nb%ORadj (95% CI)aDusts Cement dust13426.143228.70.93 (0.73, 1.18) Fiberglass dust10219.931921.21.02 (0.78, 1.33) Coal dust6312.31499.91.19 (0.86, 1.66) Soil/field dust14227.737524.91.26 (0.99, 1.61) Whey dust122.3382.50.89 (0.45, 1.77) Paper dust6813.318011.91.22 (0.89, 1.67) Wood dust14327.944529.50.95 (0.75, 1.20) Coke dust101.9583.80.53 (0.26, 1.06) Stone dust5510.717311.50.99 (0.71, 1.40) Grain Dust11722.834723.00.99 (0.76, 1.29) Sand9017.530320.10.89 (0.67, 1.16) Cardboard dust509.717011.31.01 (0.71, 1.44) Metal dust12023.436824.41.06 (0.82, 1.36)Coal Products Pitch173.3382.51.24 (0.68, 2.25) Asphalt468.91429.40.96 (0.67, 1.38) Crude petroleum305.8845.61.00 (0.64, 1.57) Tar/tar products5310.31439.51.20 (0.84, 1.69Printing Printing inks356.81348.90.90 (0.60,1.36) Printing fluid285.5966.40.93 (0.59, 1.47)Paints Paints, dyes14828.844229.31.06 (0.84, 1.33)Metals Arsenic132.5281.91.45 (0.72, 2.93) Nickel295.6855.61.11 (0.71, 1.74) Cadmium203.9553.61.07 (0.62, 1.84) Zinc387.41036.81.12 (0.75,1.67) Mercury203.9634.20.84 (0.49, 1.43) Chromium244.7583.81.33 (0.79, 2.22) Iron407.81006.61.18 (0.79, 1.77) Lead6512.718212.11.03 (0.75, 1.42) Aluminum7113.822014.61.03 (0.76, 1.40)Miscellaneous Asbestos7614.823715.70.91 (0.68, 1.21) Used motor oil11722.840026.60.89 (0.69, 1.15) Diesel exhaust fumes18335.746430.81.33 (1.06,1.67)c Cutting oils7414.427718.40.81 (0.60, 1.08) Cleaning fluids12424.241927.80.93 (0.72, 1.19) Preservatives91.7211.41.11 (0.49, 2.50) Chlorine6813.320213.41.05 (0.77, 1.43) Hair permanent solutions112.1332.20.99 (0.48, 2.04) Sour gas244.7926.10.69 (0.42, 1.12) Wood smoke12123.637124.60.95 (0.75, 1.22) Lubricants15229.647731.70.99 (0.78, 1.25) Solvents16732.551634.31.01 (0.80, 1.28) Ether519.917011.30.88 (0.62, 1.25) Mouldy grain/forage6111.917611.71.09 (0.78, 1.53) Hair dyes152.9332.21.33 (0.69, 2.52) Cyanide101.9362.40.79 (0.38, 1.63)Non-ionizing radiation Ultra Violet Light448.615110.01.06 (0.73, 1.55) Horticultural Grow lights122.3392.590.91 (0.46, 1.79) Unshielded microwaves30.6251.70.39 (0.11, 1.32)Ionizing radiation Radium122.34120.803.26 (1.38, 7.73)c Uranium122.34181.202.10 (0.97, 4.56)a all odds ratios were adjusted for age and province of residence.b n and % are given for the \"yes\" responses.c Statistically significant results are bold.Table 5 shows the results of multivariate conditional logistic regression models for the long held jobs of farmer and machinist. The variables that remained statistically significantly associated with increased risk of NHL for long held job as a farmer were personal history of another cancer and exposure to ionizing radiation (radium). The variables for the long held job as a machinist associated with increased risk of NHL were personal history of another cancer, exposure to ionizing radiation (radium) and exposure to diesel. Duration of exposure for the long held jobs of farmer and machinist were borderline significant at 5% level (p = 0.08 and p = 0.059), but there was evidence of an increase risk of NHL with longer duration of exposure.Table 5Multivariate models of the important covariates associated with NHL for long held occupations.VariableFarmerMachinistOR (95% CI)aOR (95% CI)aPersonal history of another cancer (yes)2.60 (1.83, 3.69)c2.57 (1.82, 3.65)cEver exposed to ionizing radiation (radium) (yes)3.41 (1.44, 8.11)c3.21 (1.34, 7.67)cEver exposed to diesel (yes)1.23 (0.97, 1.56)1.28 (1.02, 1.61)cDuration (reference to no exposure)<10 years0.77 (0.45, 1.30)0.73 (0.29, 1.86)10–20 years1.34 (0.54, 3.34)1.87 (0.33, 10.57)> 20 years1.47 (0.95, 2.29)2.34 (0.97, 5.68)a all odds ratios were adjusted for age and province of residence.c Statistically significant at 5% level results are bold.DiscussionOur study investigated the association between NHL and several occupations and occupational exposures. The findings revealed that two long held occupations (10 years or more), farmer and machinist, were significantly associated with increased risk of developing NHL. One of the possible explanations is that farmers and drivers might be exposed to pesticides and engine exhaust and machinists might be exposed to solvents or engine exhaust at the work place. The increased risk of NHL for farmer and machinist seen in our study is consistent with the findings from other studies [8-16].Pesticides including herbicides and insecticides have been associated with Non-Hodgkin's Lymphoma in studies of farmers, agricultural related workers, other pesticide applicators, manufacturing workers and other exposed populations [39,45]. Grain handlers exposed to pesticides, grain dusts, and organic solvents were shown a five-fold risk of NHL [46]. Our study confirms that those who held the long held job title as a farmer (farmer, farm labourer and farm managers) had 1.5 times higher risk of being diagnosed with NHL than those who held a job title from the category of non-farmer.Our results confirm previously reported associations of NHL and a personal history of cancer [47,48]. Occupational exposure to dust (wood, paper, metal etc.), coal products, paints, metal, and printing are unlikely to increase the risk of NHL, as is evident from our analysis. In contrast, Kawachi et al [49] found a significant association between working with wood and NHL. In addition, Kogevinas et al [50] found an increased risk of Lymphomas in pulp and paper workers. Ever exposure to diesel exhaust fumes is likely to increase the risk of NHL, as is evident from our analysis. Our finding is agreement for diesel exhaust fumes with Baris et al [21] and Maizlish et al [34].The mechanism of cancer induction by radiation suggested in our study is not clear. The most widely accepted hypothesis is that some of the ionizing events, which occur when radiation is absorbed in tissue, produce a change in the genes or chromosomes of one or more cells [51]. A case-referent study conducted to investigate the possible association between occupation and occupational exposures and risk of hematological malignancies showed that exposure to asbestos, hydrocarbons, fertilizer, radiation, pesticides and mineral oils were highly associated with hematological malignancies [10]. Another matched case-control study in the nuclear industry [52] found no significant excess of NHL at any radiation exposure level. Archer [51] stated that uranium mill workers appeared to have excess Lymphomas. In our study, any form of radiation exposure at work was considered. Exposure to ionizing radiation (radium) is significantly associated with increase risk of NHL, which suggests equivocal evidence of an association with NHL presented by Ron [53].There are many potential sources of non-ionizing radiation to workers. One of them is ultraviolet (UV) radiation. There is suggestive evidence that exposure to ultraviolet (UV) light, an established cause of immune suppression, may increase the risk of NHL [54-57]. The most recent epidemiologic literature suggests that there is no association or protective effect between exposure to sunlight and NHL [58-63]. Our study did not find any association between exposure to ultraviolet (UV) light with NHL.Solvents have been associated with NHL in a number of studies [64-66], including studies of rubber workers [67], aircraft maintenance workers [68], and dry cleaners [69]. In particular, benzene exposure is common in above mention occupations and this may be due to its effects on the immune system [66]. Other occupations which might involve exposure to solvents or related chemicals and which are reported as being at increased risk of NHL include those of highway workers [34], petroleum refinery employees [70-72], styrene workers [73], chemists [74,75], and chemical manufacturers [76,77]. We could not find any association between NHL and exposure to solvents, cleaning fluids, or preservatives.A major strength of this study is the large number of cases and controls from residents of six Canadian provinces. Questions were designed to obtain a complete occupational history and extensive list of potential occupational exposures. A reference pathologist validated 84% of the NHL tumours.There are, however, several limitations in this study. One of the limitations is the potential for recall bias and misclassification of pesticide exposures. Also, occupational exposures in this study were self-reported and this might also bias results. Due to budget constraints, the study was restricted to males. The response rates of 67.1% for cases and 48% for controls represent another potential limitation that could create misleading conclusions if the non-respondents differ significantly from the respondents with respect to the variables under investigation. We compared non-respondents to respondents using postal codes as an indicator of rural residence and did not find a rural bias among respondents. The most common reasons for not participating were death, change of address, and refusal for both cases and controls. Another limitation was the possibility of false-positive findings given the large number of jobs and exposures assessed.ConclusionOur results support previous findings of an association between NHL and specific job titles and occupational exposures. In our analysis, NHL was associated with personal history of cancer, exposure to diesel exhaust fumes, exposure to ionizing radiation (radium) and long held occupations such as farmer and machinist. Also, we have supportive evidence of increased risk of NHL with longer durations of exposure.AbbreviationsNHL: Non-Hodgkin's Lymphoma; ICD: International Classification of Diseases; STS: Soft Tissue Sarcoma; HD: Hodgkin's Disease; MM: Multiple Myeloma.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsCPK analyzed data and prepared the manuscript. HHM designed, coordinated the study and collect the data. JAD participated in study design, coordination, data collection and manuscript preparation. JJS participated in the design of the study and data collection. PP designed and coordinated the study as well as collected and prepared the manuscript.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2531122.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2531122",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531122\nAUTHORS: Stuart A Suttie, Reza Mofidi, Alison Howd, Gareth D Griffiths\n\nABSTRACT:\nIntroductionAxillary artery injury is a rare but severe complication of fractures of the surgical neck of the humerus.Case presentationWe present a case of axillary artery pseudoaneurysm secondary to such a fracture, in a 82-year-old white woman, presenting 10 weeks after the initial injury, successfully treated with subclavian to brachial reversed vein bypass together with simultaneous open reduction and internal fixation of the fracture. We discuss the use of a Javid™ shunt during combined upper limb revascularisation and open reduction and internal fixation of the fractured humerus.ConclusionThis case highlights the usefulness of a Javid™ shunt, over other forms of vascular shunts, in prompt restoration of blood flow to effect limb salvage. It can be considered as a temporary measure whilst awaiting definitive revascularisation which can be performed following fracture fixation.\n\nBODY:\nIntroductionProximal humeral fractures are a common injury with an incidence of approximately 5% of all fractures, with the majority being secondary to blunt trauma in an elderly population [1]. Despite the close proximity of the axillary artery and the surgical neck of humerus, injury to this artery is a rare complication of proximal humeral fractures. It is, however, associated with significant risks to both function and viability of the affected upper limb.Upper limb ischaemia secondary to such a cause requires prompt intervention to restore blood flow and subsequently treat the primary cause. Earlier reports have documented success in similar settings, using modified equipment not necessarily designed for use as an intravascular shunt [2,3].We present a case of delayed presentation of axillary artery pseudoaneurysm following proximal humeral fracture and discuss the use of a Javid™ carotid shunt (Bard carotid shunt, 17F tapered to 10F; Bard® Javid™ Carotid Shunts, Bard Ltd., Forest House, Brighton Rd., Crawley, West Sussex, UK) in maintaining vascular perfusion during open reduction and internal fixation of the fracture.Case presentationAn 82-year-old, white woman with a history of alcohol abuse, presented to the accident and emergency department with a 4-hour history of an acutely ischaemic right upper limb with motor and sensory deficit. A hard tender, pulsatile mass was palpable in the right subclavian area with significant bruising; there was a palpable right subclavian pulse with no pulses distal to this. X-ray revealed a fracture of the surgical neck of the right humerus with the humeral head abducted and externally rotated, while the humeral shaft was displaced medially (Fig. 1).Figure 1Anteroposterior view of right shoulder 10 weeks after the primary injury, revealing malalignment of fracture ends and attempts at formation of primary callus (arrow).Ten weeks previously, she had presented with a fracture of the surgical neck of the right humerus following a fall whilst under the influence of alcohol. On that occasion, sensory and motor function of the limb had been recorded to be fully intact by the medical staff in Accident and Emergency and there had been a full complement of pulses. Given she had no neuro-vascular deficit in the affected limb, the vascular surgeons were not involved initially. Under guidance of the orthopaedic surgeons, she had been treated conservatively with a collar and cuff due to her age and history of current alcohol abuse. She was to have been followed up fortnightly in the orthopaedic fracture clinic – but failed to attend after her second visit. She had no neuro-vascular deficit on follow-up. She denied any further falls or trauma to the right upper limb.The acute nature of the current presentation together with neurological compromise prompted classification as category-II acute limb ischaemia (Society for Vascular Surgery/International Society for Cardiovascular Surgery classification) [4] and urgent angiography was performed with a view to revascularisation. This revealed a pseudoaneurysm of the third part of the right axillary artery with complete occlusion of the right brachial artery distal to this (Fig. 2).Figure 2Catheter angiogram depicting pseudoaneurysm formation of third part of axillary artery with complete occlusion of the distal right brachial artery.Operative treatment was undertaken with initial exposure and control of the subclavian artery above the clavicle (Fig. 3A). Simultaneous exposure of the brachial artery in the antecubital fossa was performed and a size 3 Fogarty embolectomy catheter passed distally down the brachial artery. Both radial and ulnar arteries were found to contain thrombus which was cleared with good back flow. The proximal brachial and distal subclavian arteries were ligated in continuity. Two interconnected Javid™ shunts were inserted to carry blood flow from the subclavian to the brachial artery in order to maintain perfusion (Fig. 3B) during open reduction and internal fixation of the fractured humerus, after which a subclavian to brachial bypass was performed using reversed long saphenous vein. The fracture was temporarily stabilised using external splints to immobilize the limb whilst securing vascular continuity.Figure 3Supraclavicular exposure of the subclavian artery. (A) The phrenic nerve is retracted before the division of the scalenus anterior muscle. (B) The subclavian artery is exposed and ligated distally, with blood flow to the right arm being maintained with the aid of a Javid shunt during open reduction and internal fixation of the fracture.Postoperatively, the patient had strong radial and ulnar pulses with complete resolution of her motor and sensory dysfunction within 72 hours. Her postoperative course was uncomplicated and she was discharged on the 10th postoperative day. Early postoperative duplex scan performed at 6 weeks revealed satisfactory function of the vein graft.DiscussionDespite the fact that a significant proportion of fractures of the surgical neck of the humerus are displaced, axillary artery injuries secondary to these fractures are rare [1,4-7]. The majority affect the third part of the artery, due to its position of relative immobility, being tethered by the subscapular and thoracromial arteries [1,8]. Most of these injuries lead to thrombosis of the axillary artery and acute lower limb ischaemia [4,5,9]. Pseudoaneurysm formation of the axillary artery is rare following blunt and penetrating trauma to the shoulder, often presenting late as a pulsatile mass rather than acute limb ischaemia [1,6,7,10].Endovascular treatment with a covered stent graft has been reported previously and is the treatment of choice in patients with pseudoaneurysm of the axillary artery without upper limb ischaemia [7,11]. Due to the presence of propagating thrombus and displaced fracture requiring open reduction and internal fixation, endovascular treatment was not an option in this patient. Following proximal and distal arterial control and thrombectomy, the limb was revascularised temporarily using a Javid™ shunt, which allowed safe internal fixation of the fracture before bypass grafting. The insertion of the Javid™ shunt served to confirm the viability of the limb and adequacy of distal thrombo-embolectomy. The use of temporary shunting of peripheral vasculature in order to maintain distal vascular perfusion is rarely employed in civilian surgical practice [2,3], however, it has been gaining popularity in the management of military trauma [12-14]. Recent reports from Belfast, whereupon the use of intraluminal shunts has been advocated for the early restoration of blood flow following complex lower limb vascular injuries, have shown significant benefits in averting the incidence of fasciotomy, contractures, ischaemic nerve palsy and amputations [15]. This Belfast approach of early shunting allows for a disciplined surgical approach with adequate time for wound debridement, safe fracture fixation and optimal vascular reconstruction. Reports from Operation Iraqi Freedom suggest that vascular shunts can be used safely to bypass complex vascular injuries encountered in forward surgical units, in order to allow transfer of injured patients for definitive vascular assessment and reconstruction [12,14]. The use of vascular shunts in these circumstances was associated with very low limb amputation rates [14], even in patients in whom the shunt had thrombosed in transit [12].The Javid™ shunt has the advantage over other types of non-vascular shunt employed [2,3], in that it is specifically designed for use as a carotid artery shunt. It is manufactured out of soft, kink free material, which is tapered towards the ends which are bulbous in nature. This allows the shunt to be clamped in place around the artery, thereby providing stability whilst surgery continues. It was felt that the Javid™ shunt was superior to the Pruitt-Inahara® carotid shunt (an H-shaped carotid shunt, held in place using inflatable balloons) for this patient due to its ease of use, lack of extra lumens (which would easily be caught and cause the shunt to be dislodged), ability to interconnect two shunts and its specially designed clamps to hold the shunt in situ during extensive and vigorous mobilisation of the fractured bone during reduction and fixation. Although these shunt clamps may cause more damage to the arterial lumen than the balloon of the Pruitt-Inahara® shunt, this damaged segment of the injured artery would in turn be ligated and bypassed.ConclusionThis case highlights the usefulness of a Javid™ shunt, over other forms of shunt, in prompt restoration of blood flow to effect limb salvage. It can be considered as a temporary measure whilst awaiting definitive revascularisation which can be performed following fracture fixation.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsSAS was first assistant (subclavian exposure), carried out the literature review and constructed the manuscript. RM was first assistant (brachial exposure), photographer, carried out the literature review and drafted and editing the manuscript. AH was primary surgeon (brachial exposure), constructed the idea behind the case report, was senior editor of the manuscript (critical revisions) and gave final approval. GDG was primary surgeon (subclavian exposure), constructed the idea behind the case report, was senior editor of the manuscript (critical revisions) and gave final approval.ConsentWritten informed consent was obtained retrospectively from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2531132.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2531132",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531132\nAUTHORS: Adewale L Oyeyemi, Babatunde OA Adegoke, Adetoyeje Y Oyeyemi, Bukola M Fatudimu\n\nABSTRACT:\nBackgroundThere is overwhelming evidence of the benefits of physical activity and the physical environment is increasingly recognized as a promising determinant of physical activity participation. The influence of the environment on physical activity has not been evaluated among black Africans and no specific measure exists for assessing environmental factors related to physical activity in an African environment. The IPAQ E- module was designed to assess environmental factors for physical activity participation and was considered to be relevant to all countries regardless of the stage of economic development. The objective of this study was to assess the test- retest reliability of IPAQ E- module in an African population.MethodsOne hundred and three clinical students of a University in Nigeria were invited to participate in the reliability testing of IPAQ E- module. Sixteen of the 17- items on the environmental measure were assessed for test- retest reliability using intraclass correlation coefficient (ICC) with 95% Confidence interval (CI) overall and by gender. The measure addressed items regarding residential density, access to destinations, neighborhood infrastructures, aesthetic qualities, social environment, street connectivity and neighborhood safety.ResultsOf the total respondents, 51.5% were males and 48.5% were females. Overall, the intraclass correlation coefficient (ICC) ranged from 0.43 to 0.91. The item regarding many interesting things to look at (aesthetic) produced the overall highest reliability score (ICC = 0.91, 95% CI = 0.86 – 0.94), while the item regarding safety from crime during the day (neighborhood safety) produced the lowest overall score (ICC = 0.43, 95% CI = 0.26 – 0.57). Reliability of items on neighborhood infrastructures ranged between substantial agreement to almost perfect agreement overall (ICC = 0.66 – 0.88) and by gender (male- ICC = 0.68 – 0.90 and female- ICC = 0.63 – 0.86). The access to destination items (ICC = 0.49 – 0.74), social environment (ICC = 0.62) and street connectivity (ICC = 0.78) all had acceptable reliability overall. Meaningful differences were found between males and females on two items on neighborhood safety and one item on access to destinations.ConclusionThe test- retest of IPAQ E- module resulted in moderate to almost perfect agreement for most of the items with few meaningful differences by gender. Environmental items of physical activity in an African population exhibited reliability similar to that in other environments. These results suggest that IPAQ E- module may be a useful measure for assessing environmental correlates of physical activity among population in Africa.\n\nBODY:\nBackgroundThe overwhelming health benefits of physical activity are well documented [1,2]. There is however mounting evidence that physical activity in clearly defined context is on the decline worldwide [3] and the physical environment is increasingly being recognized as a potential and promising determinant of physical activity behaviour [4-7]. The influence of the physical environment on physical activity behaviour is currently unknown among the African population and no specific measure exists for the assessment of environmental correlates of physical activity in the African environment. However, the influence of the environment on physical activity behaviours is particularly important because physical activity occurs in specific environmental settings [8] and the environment that people build and inhabit provides potential opportunities for and barrier to engaging in a physically active lifestyle [9].Until recently, studies on environmental correlates of physical activity have focused on the narrower interpersonal and individual levels of intervention while neglecting the broader contextual framework of socioecological model [10,11]. Research in this field are strengthened by utilizing the ecological model that recognizes the multiple levels of influence on health behaviours vis- social system, public policies and the physical environment [12]. This model has potential for explaining and facilitating better understanding of the influence of the environment on physical activity behaviors than the individual focus oriented model [12,13].Sallis et al [14] highlighted the necessity of first identifying reliable and valid measures of theoretically relevant environmental variables before the influence of the environment on physical activity can be adequately evaluated. Most of the studies that have evaluated the psychometric properties of environmental measures were conducted among Caucasians, especially in the United States [14-17] and Europe [18,19], with their findings reflecting few differences in the reliability coefficients of the environmental variables. For instance, while the European studies [18,19] tend to identify items on neighborhood safety with lowest reliability coefficients, some American studies [16,17] implicated walking/cycling facilities and street/walking environments as items with the lowest reliability coefficients. There is therefore the potential possibility for the reliability of perceived environmental correlate items of physical activity to vary across countries and by cultures. Reliability studies from other continents may hence be necessary to fully compare and identify the international dimension and relevance of assessing environmental measures of physical activity behaviours.Various measures for assessing environmental correlates of physical activity are in existence [14,17,18,20]. Although, the development of these measures were based on the contextual framework of socioecological model, they are mostly lengthy, voluminous and yet to be assessed internationally. The International Physical Activity Prevalence Study (IPS) group in 2002 developed a shorter survey (IPAQ Environmental- Module), primarily for the assessment of environmental factors for bicycling and walking in the neighborhoods. The strengths of IPAQ E- module are its brevity and the inclusion of variables that have been shown to be associated with different levels of physical activity in different countries. Also, items on the E- module were considered to reflect current thinking in the field of environmental correlates of physical activity that are considered to be relevant to all countries regardless of their stage of economic development [21]. This assertion may however, need to be tested in African countries where the physical environment is distinct from that in other parts of the world.Evaluating the IPAQ E- module for reliability in an African environment is therefore necessary and may be a precursory step to identifying appropriate environmental correlates of physical activity behaviours among this population. Also, assessing the test- retest reliability of the IPAQ- E module in a cohort of African population may highlight differences, indicate cultural issues and espouse the environmental correlates that are contextually relevant to Africa. Since test- retest reliability is a useful means of assessing the reproducibility of a measure and hence the consistency and stability of an instrument over time [22], the purpose of this study was therefore to assess the test- retest reliability of the IPAQ E- module in an African population. The environment for the purpose of this study was defined as neighborhood characteristics.MethodsSampleParticipants were undergraduate clinical students of the urban based premier University in Nigeria. They were selected from an ongoing larger study on environmental and sociodemographic determinants of physical activity among students of the University. A total of 298 male and female clinical students took part in the overall baseline survey consisting of 1006 students of the University. All the clinical students that were part of the baseline survey were invited to participate in the retest of the E-module questionnaire and about 69% of them (n = 103) agreed to participate in the retest study. Participants were given the second copy approximately 7 days after the first questionnaire was returned. The questionnaire was self administered and completed in the participants' rooms with the investigator in attendance in order to reduce items' misinterpretation. Socio-demographic information such as age, sex, height, weight, ethnic group, academic programs, years of study and religion were also sought from the participants. All participants provided an informed consent and the study was approved by the University of Ibadan/University College Hospital Joint Institutional Review Committee on Human Research (UI/EC/08/0004).Measurement of environmental characteristicsSixteen self- report items from the IPAQ- Environmental Module (IPAQ E- module) designed for measuring environmental correlates of physical activity in the neighborhood were assessed for test- retest reliability in this study. The IPAQ E- module was made up of 17- environmental items that are grouped as core, recommended and optional. All core items were mandatory to be asked, while as many of the recommended items as possible should be asked in any study utilizing the E- module survey [21] [see Additional file 1]. One item from the recommended items \"How many motor vehicles in working order are there in your household?\" was not assessed due to the nature of the sample in the present study. Specifically, the study sample comprised students living in the University hostel and neighborhood was defined as the campus environment rather than their various household environments.For the purpose of this study items on the IPAQ E- module were classified into seven categories [19]: residential density (one item), access to destinations (three items), neighborhood infrastructure (five items), aesthetic qualities (one item), social environment (one item), street connectivity (one item) and neighborhood safety (four items). These items have been shown to demonstrate moderate reliability coefficients among the Caucasians [17-19]. Responses to the IPAQ E- module were based on a 4- point likert scale ranging from strongly disagree to strongly agree as well as don't know or doesn't apply options for 15 of the questions. The only item with specific response option scale was the question assessing residential density (the main type of housing in my neighborhood).Data analysisAnalysis for the test-retest reliability of each of the environmental variables was conducted overall and by gender using the one- way model intraclass correlation coefficient (ICC) along with 95% confidence interval (CI). ICC represents the total variance in the measure (subject variability and measurement error) that was due to true differences between participants (subject variability) [15]. It accounts for the variability between, rather than within the participants. The agreement levels rating suggested by Landis and Koch: 0 – 0.2 (poor), 0.2 – 0.4 (fair), 0.4 – 0.6 (moderate), 0.6 – 0.8 (substantial) and 0.8 – 1.0 (almost perfect) was used to interpret the results [19]. Descriptive statistics of mean and percentage were used to describe the socio-demographic characteristics of the participants. Statistical analyses were performed with SPSS version 10.ResultsOne hundred and three participants completed the test- retest survey. The mean age and BMI of the participants were 24.24 ± 3.55 years and 23.24 ± 4.07 kg/m2 respectively. About 51.5% were males and majority, (70.9%) was from the Yoruba ethnic group. More than half were clinical students of medicine (52.4%) and majority was of Christian religion (82.5%). The detailed general characteristics of the participants are shown in table 1.Table 1Characteristics of ParticipantsCharacteristicsMaleFemaleTotaln (%)n (%)n (%)Gender53 (51.5)50 (48.5)Age (Years) 16 – 194 (3.9)3 (2.9)7 (6.8) 20 – 2943 (41.7)44 (42.7)87 (84.5) 30 – 396 (5.8)3 (2.9)9 (8.7)BMI (kg/m2) < 18.55 (4.9)5 (4.9)10 (9.7) 18.5 – 24.933 (32.0)32 (31.1)65 (63.1) 25.0 – 29.911 (10.7)10 (9.7)21 (20.4) > 30.04 (3.9)3 (2.9)7 (6.8)Ethnic Group Ibo10 (9.7)5 (4.9)15 (14.6) Hausa0 (0)3 (2.9)3 (2.9) Yoruba33 (32.0)40 (38.8)73 (70.9) Others7 (6.8)5 (4.9)12 (11.7)Academic Programs Physiotherapy15 (14.6)6 (5.8)21 (20.4) Medicine27 (26.2)27 (26.2)54 (52.4) Dentistry11 (10.7)17 (16.5)28 (27.2)Years of Study 1st Clinical20 (19.4)23 (22.3)43 (41.7) 2nd Clinical19 (18.4)15 (14.6)34 (33.0) 3rd Clinical14 (13.6)12 (11.7)26 (25.2)Religion Islam9 (8.7)9 (8.7)18 (17.5) Christianity44 (42.7)41 (39.8)85 (82.5)The result of the test- retest reliability for all respondents and by gender is presented in table 2 [see Additional file 2]. Overall, the one week ICC ranged from 0.43 – 0.91, with the lowest value recorded for question on crime during the day and the highest value for question on many interesting things to look at while walking. By gender, the ICC ranged from 0.11 – 0.96 for males and from 0.23 – 0.87 for females. Both males and females recorded the highest ICC on residential density item (main type of housing in the neighborhood), while two items on neighborhood safety that is, traffic against bicycling and traffic against walking demonstrated the lowest ICC among males and females respectively. When exploring the gender based findings, only items that differed by at least two categories on the rating of Landis and Koch were considered as meaningful and discussed as such.Reliability of items on residential densityThe only question that assessed residential density was the main type of housing in the neighborhood. This item demonstrated almost perfect agreement overall (ICC = 0.89) and among the male participants (ICC = 0.96), but exhibited moderate agreement among the female participants (ICC = 0.56).Reliability of items on access to destinationThe reliability of items on general access to destination ranged from moderate (ICC = 0.49) to substantial agreement (ICC = 0.74) overall. The lowest reliability was exhibited by the question \"it is within 10- to- 15 minutes walk to the bus stop\" and the highest reliability by the question \"there are many shops within walking distance of university\". The three items on access to destination differed meaningfully by gender. While males demonstrated poor agreement (ICC = 0.19) on the question \"many shops are within walking distance of home\", females tend to demonstrate almost perfect agreement (ICC = 0.80) on the question. Also, the question \"many places to go within easy walking distance\" was almost perfectly reliable (ICC = 0.89) among males but only moderately reliable (ICC = 0.59) among females. The question \"it is within 10- to- 15 minutes walk to a bus stop from home\" also demonstrated higher reliability (ICC = 0.60) among males than females (ICC = 0.27).Reliability of items on neighborhood infrastructureOverall the five items pertaining to neighborhood infrastructures demonstrated substantial (ICC = 0.66) to almost perfect agreement (ICC = 0.88). The highest reliability was found for question on well maintained and unobstructed places for bicycling infrastructure. No meaningful gender differences were found in this domain except for the question on well maintained and unobstructed sidewalks in the neighborhood where higher reliability was found among males (ICC = 0.86) than among females (ICC = 0.56).Reliability of items on aesthetic qualitiesThe only question on aesthetic quality (many interesting things to look at while walking in the neighborhood) generated the highest reliability score overall (ICC = 0.91) and by female gender (ICC = 0.87). However, the reliability coefficients for both male (ICC = 0.94) and female (ICC = 0.87) fall within the same category (almost perfect).Reliability of items on social environment and street connectivityFor the social environment, seeing many people being physically active demonstrated substantial agreement (ICC = 0.62) overall with reliability somewhat higher among males (substantial agreement) than females (moderate agreement). Also, item on street connectivity (there are many four way intersections in the neighborhood) demonstrated substantial agreement (ICC = 0.78) overall with higher reliability among the male participants (almost perfect agreement) than their female counterparts (substantial agreement).Reliability of items on neighborhood safetyApart from the question on crime rate at night (ICC = 0.83), all other items on neighborhood safety demonstrated moderate reliability overall, with the lowest reliability found for question on crime rate during the day (ICC = 0.43). Two items on neighborhood safety were meaningfully different by gender. Reliability was substantial (ICC = 0.69) among males but fair (ICC = 0.23) among females when assessing question on much traffic making it difficult or unpleasant to walk in the neighborhood. However, the reliability among females had moderate agreement (ICC = 0.45) while reliability among the males had poor agreement (ICC = 0.11) when assessing question on much traffic making it difficult or unpleasant to ride a bicycle.DiscussionThis study evaluated the test- retest reliability of IPAQ E- module in an African population. The overall results indicate reliability to range from moderate agreement to almost perfect agreement. Few gender differences were observed in the reliability of some of the items on the E- module among the participants.The highest reliability coefficients were found for items on aesthetic qualities and residential density such as \"there are many interesting things to look at while walking in the neighborhood\" and \"the main type of housing in the neighborhood\". Items on neighborhood safety such as \"crime rate make it unsafe to go on walk during the day\" and \"so much traffic on the street makes it difficult or unpleasant to walk\" demonstrated low reliability coefficient. This result reflects the stability of question pertaining to more objective features of the environment as aesthetic (trees, flowers, landscaping view etc) and types of housing (residential, office buildings, apartments etc) than subjective environmental features such as crime and safety that are easily overtaken by time and events. It is possible that participants in this study found it more difficult to subjectively assess crime rate and safety in their neighborhood, thereby reducing the reliability of the items. There is substantial interest in perceived crime as a correlate of physical activity behaviour and studies to date have produced inconsistent results on the association between perceived crime and physical activity [7,13,18,23]. Future studies may need to utilize more objective measures of crime and safety to identify the important relationship that exists between neighborhood safety and physical activity behaviours.In a similar study [19] in Sweden, the main type of housing in the neighborhood was identified as the item with the highest reliability coefficient and neighborhood safety item \"the crime rate makes it unsafe to go on walk during the day\" demonstrated the lowest reliability coefficient. Also, in an American study [16], items on residential density were found to demonstrate high reliability while lower reliability was found for the question on unsafe walking during the day due to crime. Somewhat consistent with the higher reliability found for the item on aesthetic quality in this study, the question on many interesting things to look at while walking was found to have the highest reliability coefficient within the domain of items assessing neighborhood aesthetic [16]. The replication of these findings in an African population supports an international assumption that environmental variables pertaining to objective features like aesthetic qualities and housing type may be more reliable correlates of physical activity than subjective features like perceived crime and traffic.In this study, environmental items on neighborhood infrastructures demonstrated good reliability coefficients that ranged from substantial agreement to almost perfect agreement. This finding may suggest stability of items assessing neighborhood infrastructures. This is likely because participants in this study lived in an environment where infrastructures like sidewalks and recreational facilities can readily be perceived as available but infrastructures like bicycle facilities may not be readily perceived as available. This can increase consistency and stabilize variation between responses thereby influencing good reproducibility of the items assessing neighborhood infrastructures. Similarly to the finding in the present study, Alexander et al [19] found substantial agreement in the reliability of all items on neighborhood infrastructures, while Brownson et al [16] found reliability of items assessing infrastructures for walking and cycling to vary from moderate agreement to substantial agreement. Somewhat consistently, another study from the United States [17] reported moderate agreement for walking and cycling facilities but found the subscale to demonstrate the lowest reliability when compared with other subscales. Also, in a Belgium study [18]; items on availability of sidewalks and bike lanes demonstrated almost perfect agreement. These findings suggest that variables assessing neighborhood infrastructures are ubiquitously reliable regardless of the geographical locations.Several studies have implicated access to destination as an important correlate of physical activity [6,13,24]. In this study, the reliability of items on access to destination ranged from moderate agreement for the question \"it is within 10- to -15 minutes walk to a transit/bus stop\" to substantial agreement for the question \"there are many places to go within easy walking distance\". The lack of specificity in the time period given for the question \"it is within 10 to 15 minutes walk to a transit stop\" may reduce consistency between responses thereby lowering the reliability of the item. A previous study [18] that used a specific time and narrower definition of the neighborhood reported higher reliability coefficient for the same item. Also, the non attribution of time frame to the question \"many places to go within walking distance\" may explain its higher reliability when compared to other questions with time dimension. Consistently, a reliability study of similar sample size [20] reported comparable reliability coefficient for the question many places to go within walking distance (ICC = 0.63) to that of this study (ICC = 0.74). Similarly, the Sweden study [19] found reliability to range between (0.46 – 0.81) for items on access to destination, with particularly lower and higher reliability scores for the questions \"it is within 10- to- 15 minutes walk to a transit stop from my house and \"there are many places to go within easy walking distance\" respectively. Also, items on land use mix- access (store within walking distance and easy walking to transit stop) and land use mix diversity (how long from home to get to business or convenience facilities) were found to demonstrate substantial to almost perfect reliability in an American study [17]. Replicating similar findings in an African population suggests an overwhelming importance of land use mix access as a reliable and viable environmental correlate of physical activity.Substantial agreement was also found for the only item on social environment (seeing many people being physically active) and street connectivity (many four way intersections in the neighborhood). Similarly, the Sweden study [19] found substantial reliability for the only item on street connectivity but lower (moderate) reliability coefficient for the item pertaining to seeing many people being physically active in the neighborhood. However, an American study [16] found a somewhat lower reliability (ICC = 0.51) for the question on many four way intersections when compared to other studies. This was however not substantial since the value still falls within the moderate reliability found in these other studies [17,18].Like the previous studies [19,20] that have assessed gender differences in the test- retest reliability of environmental measures of physical activity, few items demonstrated meaningful gender differences in the present study. Reliability was meaningfully higher among males than females on questions involving residential density, many places to go within easy walking distance, it is within 10- to- 15 minutes walk to the transit/bus stop and much traffic making it difficult or unpleasant to walk in the neighborhood. However, females had meaningfully higher reliability score on the questions \"many shops within walking distance\" and \"so much traffic making it difficult or unpleasant to ride bicycle in the neighborhood\" than males. Since males generally do not engage in shopping, it is plausible for them not to be consistent overtime in response to question on awareness of shops within walking distance of the university as compared to the females who may always be aware of shops because they regularly do shopping and hence answered more consistently, which consequently resulted in the meaningfully higher reliability of the item among females. While it may be difficult to draw any definite conclusion from these findings, it reflects the potential influence of gender on the reliability of some environmental correlate items. This is likely because evidence is now emerging that gender may exert a moderator link between perceived environment and physical activity behaviours [6,18,23].LimitationsThis study has some important limitations. The generalization of this study is limited by the nature of the sample used which comprised clinical students of the University with potential higher comprehension and recall ability than may be found in the general population. The findings should therefore be interpreted with caution among other blacks of diverse educational level. Also, lower variation may exist in the limited environmental features available on campus than in the general community and this may lower reliability for the assessed environmental items in this study.Like all other studies involving recall, it is possible for participants in the present study to give vague rather than factual responses to perceived environmental features in their neighborhood, thereby affecting results and inferences from this study. Also, though quite unlikely because of the short time frame, actual changes might have occurred between the retest surveys thereby reducing the observed reliability coefficients. The 95% confidence intervals for some of the reliability estimates, especially for the gender based findings were not precise thus suggesting the possibility of a low sample size.The non utilization of objective environmental measures that would have served as the criterion for assessing the validity of IPAQ E- module in this study may constitute a limitation. Previous studies [18,25] have observed actual differences between objective and subjective data of environmental measures of physical activity. However, since no data presently exist in this field among the African population, the use of self- report measures of the environment may still hold sway for the time being in the African environment. This however does not suggest the non-importance of evaluating both subjective and objective environmental measures even at this early stage of this research type in African populations.ConclusionThis study has for the first time provided evidence on the psychometric properties of items on an environmental measure of physical activity in an African population. The test- retest reliability of IPAQ E- module ranged from moderate agreement to almost perfect agreement with few meaningful differences by gender. The reliability coefficients of environmental items among this population were mostly similar to that in other environments. Items on IPAQ E- module is therefore promising and may be useful for assessing environmental correlates of physical activity among the black population in Africa. Future studies should consider testing IPAQ E-module along with more objective environmental measures in a diverse African population.Authors' contributionsALO conceived and designed the study, analyzed and interpreted the data, drafted the manuscript and gave approval for the final version. BOA was involved in study design, interpretation of data and drafting of the manuscript. AYO was involved in study design and drafting of the manuscript. BMF was involved in data acquisition and read the manuscript for final approval. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2531173.json

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+{
+    "id": "PMC2531173",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531173\nAUTHORS: Jagdeep S Thakur, CGS Chauhan, Vijay K Diwana, Anamika Thakur\n\nABSTRACT:\nPeri-anal contracture lead to intestinal obstruction whenever there is involvement of anal orifice. In this case anus and peri-anal skin up to two cm was normal; however both gluteal folds were fused because of post burn scar leaving a very small opening which lead to faecal impaction and sub acute intestinal obstruction.\n\nBODY:\nBackgroundManagement of the burn patient is the most challenging condition for the medical staff as the fate of the patient depends on the quality of the management provided during hospital stay and after discharge. Even if the patient recovers from the burn injuries, the development of the deformities overshadow the earlier management. This post burn reconstructive surgery and physiotherapy consultation needs to be made compulsory in the burn units.Case reportA two and half year old male child was admitted with complaint of progressive difficulty in passing stools along with progressive distension of abdomen, for last one year. There was history of vomiting, off and on for the last fortnight. Patient had history of sustaining 10% thermal burns over perineum, gluteal region and left foot about one and half year back.On examination, there was mild distension of abdomen and occasional visible peristalsis movement with exaggerated bowel sounds. Examination of perineum showed that both the gluteal folds were fused because of post burn scar and there was a small opening approximately three mm in diameter in the centre (Figure 1). Rectal examination could not be carried out through this opening. The general physical and other systemic examination was normal.Figure 1Peri-anal post burn contracture obstructing normal view of anal area.The blood investigations were normal and x-ray abdomen showed few air fluid levels. The child was operated under general anaesthesia. The contracture was released and both gluteal folds were separated. Raw area was grafted with split thickness skin graft. When contracture was released, it was found that anal verge along with peri-anal skin up to two cm was normal (Figure 2). It was fusion of gluteal folds due to post burn scar which led to sub acute intestinal obstruction. There was faecal impaction in the rectum. Post operative recovery was uneventful and graft was well taken.Figure 2Intraoperative view with skin graft in place.DiscussionIn the review of literature (Medline search), there are number of publications on the perineal burn and its management in the children [1-4]. As compared to this, we have found only one publication on perineal contracture leading to anal stricture and mega rectum in a three years old child [3]. In comparison to this report, in our case there was no involvement of anus or rectum. The intestinal obstruction was due to the post burn contracture in the gluteal fold which lead to the obstruction beyond the anus. This contracture was released and patient had complete recovery without any sequel. One more interesting fact in this case was that the patient sustained burn injury in gluteal area by sitting on the 'Chullah' (Figure 3), an earthen made stove, in which wood is used as fuel, a very common practice for the cooking in rural area of our country.Figure 3'Chullah' a traditional stove.ConclusionAlthough perineal and gluteal burns are rare even in the rural areas of our country, as people are now using natural gases for the cooking etc but this rare case report emphasises on the critical burn care, post burn care, physiotherapy and regular follow up to the hospital E. Ye [4] has also given emphasises on the meticulous preoperative and post operative care in patients with chronic obstruction due to peri-anal contractures.ConsentThe written informed consent of the patient has been obtained for the publication of this case report and accompanied images.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsJST has designed and written the article and is the principal contributor, CGS was involved with the management of the patient, conception, design and review of the article, VKD was involved in the management of the patient, conception and critical review of the article, AT was involved in acquisition of the data, review of the literature and critical review of the article. All the authors have read and given final approval for this article\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2531174.json

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+{
+    "id": "PMC2531174",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531174\nAUTHORS: Sandeep Shrivastava, Shishir P Nawghare, Yogesh Kolwadkar, Pradeep Singh\n\nABSTRACT:\nBackgroundWe present a case of a 35 yrs old female who presented with swelling over her forearm. This is a rare case of a giant cell tumour in a nonepiphyseal region.MethodsCase report and presentation of clinical, radiological and histological data on single case of giant cell tumour of diaphysis of radius.ResultsAlthough age, clinical features and radiological features are helpful, it is still the histology that helps to clinch the diagnosis.ConclusionA thorough literature search and an exhaustive online search using various search engines revealed seven reported cases of giant cell tumours in the diaphysis of long bones. We reiterate the fact that irrespective of the location, a giant cell tumour should be diagnosed based on its histology.\n\nBODY:\nIntroductionGiant cell tumour is a benign but locally aggressive tumour. The classification and definition of giant cell lesions was first proposed by Jaffe and Lichtenstein[1]. 20% of all benign bone tumours and 5% of all tumours are giant cell tumours. It is more common in young adults between 20 and 40 years of age [2-4]. It is more common in females with the rate of growth enhanced in pregnancy[5].Appearance before epiphyseal plate closure is rare[2,6,7]. It occurs commonly in the distal femur, the proximal tibia, the distal radius and the sacrum[2-4]. Giant cell tumours (GCT) usually prefers the epiphyses of long bones. The involvement of the metaphysis or diaphysis without epiphyseal extension is quite uncommon[1]. Often the tumour extends to the articular subchondral bone, However it seldom crosses the joint or its capsule. If the GCT appears prior to epiphyseal, it is likely to be found in the metaphysis[8,9]. A diaphyseal GCT is almost unheard of A literature search brought forth very few reported cases[10-14]. This is perhaps the eighth case of diaphyseal GCT reported in the literature. It recurs from time to time and rates between 25–50% have been reported[2,6,15]. In very rare cases, a malignant change may occur[14,16,17]. Taking this into account, it is essential that a correct diagnosis of GCT should be made. It is essential that we are aware of the rare existence of giant-cell tumours in areas other than the epiphysis. We may miss a few if we are not [10].Case ReportA 35 years old Asian school teacher was admitted in our hospital with a complaint of swelling over her left forearm. The swelling had increased gradually over the preceding year. She also complained of occasional pain over the inside of her forearm. She did not sustain any kind of trauma or suffer from any fever in her last few months. Examination revealed a diffuse fusiform swelling over the middle third and outer aspect of her left forearm. The overlying skin was tense. No signs of inflammation were visible. On palpation, there was tenderness over the swelling, especially over the lateral aspect. The swelling was soft in consistency with a feeling of 'egg shell crackling'. Movement at all the joints was full in range and was painless. There was no neurovascular deficit. The calcium, phosphorus and parathyroid levels in the serum were within normal limits. A radiograph of the forearm showed an expansile lesion of 10 cms × 5 cms over the middle third of the radius (Fig 1). The lesion was lytic and ballooned. The cortical margins were thinned out and breached. The chest radiograph was normal. A fine needle aspiration biopsy was done.Figure 1Radiograph of diaphyseal giant cell tumour.To our surprise, it was reported as a giant cell tumour It was decided to excise the tumour. At surgery, the tumour was reddish brown, ovoid in shape and soft in consistency. Frozen section was done to know the extent. It extended from the metaphyseal-diaphyseal junction area of the distal radius to the proximal fourth. It was removed cleanly. The ulna was centralised over the third metacarpal and the wrist was arthrodesed with a dynamic compression plate (Fig 2).Figure 2Radiograph-two years postoperative.Histology revealed large number of scattered giant cells with centrally placed nuclei. The tumor was composed of plump spindle shaped cells. It was also reported as a giant cell tumour. The tumour did not recur two years after the surgery and the patient is in good health.DiscussionMost GCTs are present in the epiphyseal or epimetaphyseal end of the long bones.If the epiphysis is not involved, a diagnosis of GCT is dubious. The radiographic findings are helpful but cannot clinch the diagnosis. Histological examination is still the gold standard for diagnosis.To distinguish metaphyseal and diaphyseal GCT from other lesions is a challenge for the pathologist. Several lesions like aneurysmal bone cyst, giant cell-rich osteosarcoma, fibrous cortical defect, solitary (unicameral) bone cyst, and giant cell lesion of hyperparathyroidism (brown tumour) are more common in these sites than are true GCT. This extensive range of lesions with the exception of giant cell lesion of hyperparathyroidism, usually appears in the first two decades of life.Aneurysmal bone cyst is clinically benign It commonly appears in the metaphysis. It contains prominent blood-filled spaces. The more solid zones within them exhibit fibrogenesis and osteoid trabeculae[12]. The stroma between the spaces contains hemosiderin laden macrophages, chronic inflammatory cells and broad seams of reactive osteoid[5]. Multinucleated giant cells are often conspicuous. Mitoses may be numerous, but anaplasia is absent.Osteosarcoma is a lesion in the metaphysis and contains numerous benign giant cells. The stroma reveals cells with ananplasia and irregular size and shape. Also presence of cartilage is not uncommon[12].Fibrous cortical defect is a benign lesion which regresses spontaneously. Radiology shows a characteristic eccentric zone of rarefaction with well-defined scalloped margins[12]. The microscopic picture reveals a mixture of collagen and fibroblasts with irregular cluster of histiocytes filled with lipid and hemosiderin. Multinucleated giant cells may be found[5].A simple bone cyst generally touches the epiphyseal growth plate. It is benign lesion and shows radiolucence with fine trabeculation. It contains fibrous tissue with few giant cells[12].A giant cell lesion of hyperparathyroidism appears in the metaphysis. Many giant cells scattered in a fibrogenic stroma may be present. A serum hypercalcemia and hypophosphatemia is characteristic[12].Giant-cell reparative granulomas demonstrate an appearance which suggests previous injury and inflammation with subsequent fibrosis. These lesions characteristically have an appearance which suggests previous injury and inflammation with subsequent fibrosis. Giant cells are found in the vicinity of old areas of hemorrhage, though not dispersed throughout the lesion. Mandible is a common site for these lesions[10].The case we have reported did not contain areas of hemorrhage, hemosiderin pigment, osteoid, bone, or significant amounts of collagen. The lesion also did not have the fibrotic, scarred appearance of a fibrous cortical defect, reparative granuloma, or brown tumor of hyperthyroidism. The patient had no detectable parathyroid dysfunction. The clinical and histological features of other osseous lesions which may contain giant cells were not present.The patient's age, the location of the lesion, its roentgenographic appearance, and the gross and microscopic appearances are crucial to unravel the mystery of an osseus lesion. However, the final diagnosis depends on the tumour's histological appearance only[10]. As Jaffe has mentioned 'A bone lesion may be uncharacteristic in all other respects, but if it exhibits the cytological pattern of a giant cell tumour, it should be recognised as a GCT' [18].Competing interestsThe authors declare that they have no competing interests.Authors' contributionsSS drafted the manuscript and operated on the patient. SPN, YK and PS drafted the manuscript, performed a literature search and participated in the management. All the authors read and approved the final manuscript.ConsentWritten informed consent was obtained from the patient for publication of this case report and accompanying images.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2531177.json

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+{
+    "id": "PMC2531177",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531177\nAUTHORS: David R Johnson, Anna M Ziersch, Teresa Burgess\n\nABSTRACT:\nIntroductionMany refugees arrive in Australia with complex health needs. In South Australia (SA), providing initial health care to refugees is the responsibility of General Practitioners (GPs) in private practice. Their capacity to perform this work effectively for current newly arrived refugees is uncertain. The aim of this study was to document the challenges faced by GPs in private practice in SA when providing initial care to refugees and to discuss the implications of this for policy relating to optimising health care services for refugees.MethodsSemi-structured interviews with twelve GPs in private practice and three Medical Directors of Divisions of General Practice. Using a template analysis approach the interviews were coded and analysed thematically.ResultsMultiple challenges providing care to refugees were found including those related to: (1) refugee health issues; (2) the GP-refugee interaction; and (3) the structure of general practice. The Divisions also reported challenges assisting GPs to provide effective care related to a lack of funding and awareness of which GPs required support. Although respondents suggested a number of ways that GPs could be assisted to provide better initial care to refugees, strong support was voiced for the initial care of refugees to be provided via a specialist refugee health service.ConclusionGPs in this study were under-resourced, at both an individual GP level as well as a structural level, to provide effective initial care for refugees. In SA, there are likely to be a number of challenges attempting to increase the capacity of GPs in private practice to provide initial care. An alternative model is for refugees with multiple and complex health care needs as well as those with significant resettlement challenges to receive initial health care via the existing specialist refugee health service in Adelaide.\n\nBODY:\nBackgroundUnder its Humanitarian program Australia accepts approximately 13,000 refugees each year [1]. Most of these will have experienced torture and trauma, forced migration and family separation, commonly the result of prolonged war or civil conflict [2,3]. Many will have had limited or disrupted access to medical care and will have spent long periods in refugee camps in environments that are extremely unsafe, where sanitation is poor and where access to safe drinking water and a nutritional diet is limited [4]. As a result, refugees often arrive in Australia with a complex mix of physical and mental health problems many of which are rarely seen in Australia [5-7]. The recent focus of the Australian Humanitarian Program has been to resettle those who have endured protracted refugee situations and who have originated from regions of very low socio-economic development. This has seen a large increase in the number of African refugees arrivals over the past 10 years – from 16% of the total annual intake in 1998/99 [8] reaching a peak of 70% in 2004/05 [1]. SA, which receives approximately 10% of the intake, has seen a similar change in the pattern of refugee resettlement [9]. Evidence is emerging that current refugee arrivals experience significantly poorer health status in addition to even greater resettlement challenges [10-17].Despite the potential for high levels of morbidity, refugees undergo only a limited health assessment prior to their arrival in Australia. For the majority this includes a medical examination, a Chest X-ray for those 11 years and over and an HIV test for those 15 years and over [18]. Although more recently some refugees have received an additional health check a few days before departure, this is primarily to assess their 'fitness to fly' and a number of high prevalence infectious diseases and nutritional deficiencies are not included in this assessment [19]. As a result, refugees will often arrive with health conditions not previously identified [20] and there is a general consensus nationally[2,11,19,21] that all newly arrived refugees should undergo a voluntary comprehensive health assessment. Such an assessment would focus both on psychological as well as physical health needs in addition to providing information about illness prevention and health promotion activities and an introduction to the Australian health care system.Until recently in SA, the State funded refugee health service along with two community health centres (CHCs) with specific refugee health expertise, performed comprehensive health assessments on a large proportion of newly arrived refugees to SA. With changes to the Department of Immigration and Citizenship (DIAC) funded Integrated Humanitarian Settlement Service (IHSS) contract in 2005, the responsibility of providing this initial care in SA was passed to General Practitioners (GPs) in private practice. Whilst this is the path taken in SA, each Australian state and territory has a different model for the provision of initial health care services with varying levels of involvement of specialist refugee and community health services [19], although GPs in private practice still provide a large proportion of this care. Recognising that GPs require extra assistance to do this work, the Federal Government introduced a new Medicare item number in May 2006 to better remunerate GPs who perform initial refugee health assessments.There has been surprisingly little written documenting the experiences of GPs who provide initial care to refugees, both in Australia and overseas, including the challenges they might face and hence their capacity to do this work is uncertain. Some general review articles have been written by health practitioners based on their own experiences providing health care to refugees and challenges listed include those related to language, [3,22] the time consuming nature of the work [22,23], cultural differences [22,24] and the special health care needs of refugees [24]. Additional challenges found in a limited number of empirical studies overseas include GPs being unaware what previous screening or treatments refugees had undergone [25,26], that there were a lack of targeted services for refugees, [25] that refugees had greater heath care needs compared to non-refugees and that GPs lacked familiarity with the management of conditions unique to refugees [27].There are limitations, however, in the applicability of these studies to the Australian context given differing national health systems and special service entitlements for refugees and the fact that it was unclear to what extent these studies related to the experiences of GPs providing care to refugees from similar backgrounds to those currently arriving in Australia. A review of the Australian literature found only one published study addressing this issue. In a report to the Victorian Department of Human Services, the Victorian Foundation for Survivors of Torture (VFST), drawing on their experiences as well as interviews with 19 GPs performing initial health assessments for newly arrived refugees in Melbourne, identified a number of similar challenges for GPs to those already listed [2]. Additional challenges included those related to the Australian Medicare fee-for-service system of remuneration which provided only a limited incentive for GPs to offer longer consultations or participate in 'extra consultation' activities often required when providing care to refugees. Challenges were also encountered relating to problems refugees experienced navigating the health system (eg. participating in follow-up GP care or attending referrals) and GPs faced difficulties sustaining their involvement because of the stressful nature of the work.Whilst the VFST study provides valuable insight into the experiences of GPs in the Australian context, much of the initial health care for refugees in Victoria is performed by GPs working in State funded community health centres where they are more likely to be salaried and have better access to supports to assist them to manage patients with multiple and complex health needs. By contrast, in SA all newly arrived refugees are referred directly to GPs in private practice for initial health care and there has been no research to date assessing their experiences providing care to refugees in SA.Given that current refugee arrivals are likely to carry a greater disease burden combined with the recent increased responsibility of GPs in private practice in SA to provide initial care, the aims of this study were: (i) to document the existence and nature of challenges for GPs who do this work in SA, (ii) to explore the ways in which these challenges could be reduced and (iii) to discuss the policy implications of this in relation to optimising the initial health care for refugees.MethodsDesign and participantsGiven that the nature of this study was exploratory, a qualitative approach was taken in order to gain a deeper understanding of the challenges faced by GPs in private practice when providing care to refugees. Semi-structured interviews were conducted with 12 GPs providing care to refugees in private practice as well as the Medical Directors of three of the Divisions of General Practice in metropolitan Adelaide with high levels of current or proposed refugee settlement. The study was approved by the University of Adelaide Human Research Ethics Committee.To recruit GPs, potential participants were identified via a database of GPs (held by the state funded refugee health service) who were either currently accepting or had accepted refugee referrals in the past. One of the authors (DJ) also used his personal knowledge of GPs known to provide care to refugees through his previous work at the SA specialist refugee health service and through formal and informal networks in the refugee health sector. Additional GPs were also identified following the Division interviews. An introductory letter with a fax-back reply was sent to 77 potential GP participants. After the initial mail out, six GPs agreed to participate. Follow-up phone calls were subsequently made to another ten GPs in order to recruit the remaining six GP participants. GPs were recruited from most regions of the Adelaide metropolitan area although there were none from the southern Adelaide region (resettlement of refugees to this region had only occurred relatively recently). The twelve GPs represented eight separate practices – two groups of three GPs were recruited from the same practices. Two thirds of participants had longstanding involvement in providing care to refugees whereas the remainder had become more recently involved with increasing numbers of African refugees resettling close to their practices. Whilst African refugees made up a large proportion of newly arrived refugees seen in the past twelve months, GP participants also reported providing care to large numbers of refugees from the Middle East as well as a small number of refugees from the Former Yugoslavia.To recruit the Medical Directors of Divisions, five were contacted by email with two agreeing to participate. Follow-up telephone calls led to the recruitment of one further Division.Data collectionGiven that a number of potential challenges were identified from the literature as well as there being much anecdotal evidence of challenges for GPs to do this work, a semi-structured interview format was chosen to examine these specifically whilst at the same time allowing any previously unknown challenges to emerge. Different interview schedules were used for the GPs and the Divisions respectively. Although the questions for each group focused on the broad aims of the study they addressed slightly different aspects of the issue. The GP interviews generally explored the challenges GPs face when working with refugees whilst the Division interviews focused on the current or potential role of Divisions to support GPs in private to work with refugees and well as the identification of potential structural impediments for GPs doing this work.The interviews were conducted between April 2006 and July 2006. Each participant was interviewed once with interviews ranging from 40 to 70 minutes. Individual interviews were conducted with nine of the GP participants and a small group interview was conducted with the remaining three GPs. The three Divisions were each interviewed individually. The interviews were tape recorded and transcribed verbatim. In both the GP and Division interviews data saturation was reached.Analysis and reporting of resultsA template analysis approach was adopted [28,29] where a coding template was developed which included a priori themes in addition to new themes identified from initial reading and analysis of the transcripts. Final thematic templates for both the GP and Division transcripts were agreed upon by the Project Team and then all data was coded according to these themes, with DJ undertaking the bulk of the coding. Two transcripts were also independently coded by the other members of the Project Team. Following this, comparisons were made and a consensus reached on how the remaining data was to be coded. Coding numbers were randomly assigned to protect the confidentiality of the participants where direct quotes were reported in the results.ResultsGPs in this study reported a range of challenges when providing care to refugees. In many cases these challenges were explicitly linked to performing initial assessments whilst at other times GPs spoke of challenges in the broader context of providing care to refugees – but which can be assumed to be operating when providing initial care. The challenges fell into three main categories: (i) refugee health issues; (ii) GP/refugee interaction; and (iii) the structure of general practice. There was a great deal of overlap, however, between these categories and a very strong theme to emerge was not having enough time to do the work required which related to any issue that made a consultation with a refugee longer. Further, these challenges did not appear to be related to how long GPs had been providing care to refugees or to the intensity of their involvement other than that more experienced GPs had a greater awareness of available interpreter services. The Divisions also reported challenges assisting GPs to provide care relating to a limited awareness of refugee numbers settling in their divisions and which GPs needed extra support as well as a lack of specific Commonwealth funding to do this work. Finally, whilst participants suggested ways these challenges could be reduced, overall strong support was provided for initial health care to be provided via a specialist health service.Challenges for GPsRefugee health issuesChallenges for GPs providing health care to refugees that related to refugee health issues included GP knowledge of previous health assessments, GP awareness of and experience managing health conditions unique to refugees, and the multiple and complex nature of refugee health conditions.GP knowledge of previous health assessmentsA number of GPs and Divisions expressed uncertainty regarding what health assessments refugees had received prior to arrival in Australia:I don't know if there is some sort of system that they go through, or some sort of protocol that they, medically, have to go through before they are granted visas... (Dr 1)There was also uncertainty regarding what health assessments were carried out after arrival in Australia with some GPs assuming that the MHS still performed this work. Uncertainty regarding previous assessments did not relate to how long GPs had been providing care to refugees.For some GPs this resulted in confusion over their role in detecting and managing health conditions unique to refugees:So we have got the clinical exotica; we have got very little understanding of what has happened to these people before and where the responsibility stops and starts for who should be following up all these things. (Dr 7)GP awareness of and experience managing health conditions unique to refugeesOnly one GP reported using guidelines to assist screening for exotic conditions. It was likely that many conditions unique to refugees were not being detected as indicated by one GP:I haven't personally come across anything unusual that would be something that was quite rare... I'm sure I will. I'm sure I have probably missed heaps, too. Slipped through that I haven't seen or recognised. (Dr 1)Concern was expressed, however, that refugee screening guidelines would just be another one of many such guidelines that GPs were expected to know about and follow.Even if GPs were detecting conditions unique to refugees, there was concern that they did not necessarily have the experience to manage them:I guess it is out of our comfort zone, because our medical experience doesn't include the exotic illnesses that they front up with... (Dr 7)Again, these challenges were not dependent on how long GPs had been providing care to refugees as many of these challenges related to more recently arrived African refugees.The expectation that GPs develop this expertise was also questioned as this was seen to compete with what was thought to be the important broader generalist role of general practice:...we are supposed to be highly trained now in mental health and refugee health...when actually we are general practice. We are not specialty people...I think it is important that we stay that way, because you start going down into specialty areas too much and you start to miss the bigger picture... (Dr 1)In terms of mental health problems, most GPs were aware of previous traumatic experiences of refugees and that combined with the stresses of resettlement meant that psychological problems often resulted. This also meant, however, that the time it took to build rapport and trust when seeing a refugee was far greater than with a non-refugee patient and this affected the ability to gather information about their background and past medical history.The multiple and complex nature of refugee health conditionsMost GPs described providing care to refugees as demanding, with refugees more likely to have multiple and complex health needs:...usually the refugees or migrants that I have seen have got multiple needs. It is usually not just one simple thing. (Dr 10)This often meant the nature of the work was time consuming:Often at the beginning there are so many issues to get through that I think it takes quite a number of long consultations before you really even have a clear idea about who this person is and what their experiences are. To get all the health issues on the table, I think, takes a really long time. (Dr 11)GP-refugee interactionA number of challenges relating to the interaction between GP and refugee were raised including issues related to culture and language as well as refugee knowledge of the Australian health care system.Issues related to cultureA number of GPs observed that refugees often had a different understanding of disease causation when compared to a Western model:...the way people behave around their health and their illness is very culturally determined. To try and understand what is going on, I can't just impose my framework, because the way they will express themselves is really different about what they are feeling. (Dr 11)As a result, GPs reported having to spend much longer than normal explaining Western health concepts to refugees. This included screening activities such as Pap smears and organising referrals for mammograms, when giving a diagnosis of an illness such as Hepatitis B or when referring refugees for pathology tests. Other challenges attributed to cultural differences included uncertainty over cultural appropriateness of examination, gender related issues such as decision-making over birth control and gaining consent for invasive procedures:...one woman came in and consented to a Pap-smear and thankfully, we managed to get around it that she actually had one before and was okay to do it, but I thought if she had never had one, how was I going to explain to this woman what I was going to do. (Dr 8)Different naming practices also sometimes presented challenges in locating the correct patient file. Although GPs with longer standing involvement in refugee care may have been more aware of cultural differences, all GPs reported challenges related to this issue.Issues related to languageA number of challenges relating to language resulted from the need to use interpreters. These included not being able to adequately provide explanations via an interpreter, difficulties dealing with mental health problems and the extra time required to both conduct a consultation with an interpreter as well as organise an interpreter when one was not pre-booked. Communication challenges were also experienced when contacting refugee patients for follow up of test results or to book an appointment. Although most GPs were aware of the need to use an interpreter when refugees were not fluent in English, those GPs with more recent involvement in refugee care were more likely to be unfamiliar with all the services offered by the Commonwealth Translating Interpreting Service (TIS), such as the doctor's priority phone line (where an interpreter can usually be made available on demand):The times that I have needed it they have been – appointments have been booked well in advance. How do you book an interpreter when someone rings up at lunchtime and sees you two hours later for something that is minor or insignificant? (Dr 1)One GP was confused about who paid for TIS believing that the practice was billed for an interpreting service when it was booked and the patient did not attend the appointment. A number of GPs also talked about difficulties contacting refugee patients for follow up of test results or to book an appointment:Communication, when you know they can't speak English, so you can't phone them, and when you know that they are quite a mobile group of people, so that when you send a letter to their address, they might have moved on. (Dr 6)Refugee knowledge of the Australian healthcare systemAlmost all GPs mentioned difficulties resulting from refugees' lack of familiarity with the Australian health care system. This related to missed appointments, which meant no remuneration for GPs, or refugees arriving late:That is a difficult problem with them... they will turn up really late for an appointment with no sort of seeming reference to the timeslot that they were given... it does sometimes make it difficult for us if we are then on the back foot for the rest of the session. (Dr 1)GPs also reported that refugees' lack of understanding of the Australian health system resulted in challenges for GPs when they were referring refugees to other agencies or specialists and also when writing prescriptions. As a result GPs reported spending more time providing explanations about how the health system worked:I take more time with them...Because the health care system here is much different... I try to make it easier for them to understand the system and how it works here. (Dr 5)Structure of general practiceA number of challenges relating to the structure of general practice were identified including GP workforce shortages, a lack of organised referral pathways for refugees to general practice as well as a lack of remuneration and infrastructure support required to perform initial assessments.GP workforce shortagesAs a result of the demand for GP services outstripping supply in some regions of Adelaide, providing appointments for any new patients, whether they were refugees or not, was often difficult. Three GPs in this study, with high loads of patients with complex health care needs including refugees, had closed their books to new patients and another GP described potential difficulties accepting new patients:We are having trouble accepting new patients full stop... freely accepting new patients irrespective of whether they are a refugee or not, it is difficult to actually accommodate everybody. (Dr 1)Further, as highlighted by one Division, with GP shortages most acute in socioeconomically disadvantaged areas, refugees were more likely to be affected given that they are often settled in areas where housing was cheaper:The cheaper areas for housing [are] where the workforce is the worst, so you can end up in this vicious circle where the practices go \"Ah, we are closing our books, we are just not seeing anyone new\". (Div 2)Referral systemsOverall GPs reported that there was usually no clear referral pathway for refugees to private general practice. This was perceived as a problem because it meant that GPs were not able to control the numbers of refugee patients they saw when there were limits to the amount of work they could do with patients with multiple and complex needs such as refugees:... because it is primary care you are expected to just take everybody that walks through the door. That doesn't work... lots and lots of agencies would like to refer here. We have to somehow prevent ourselves from drowning. (Dr 11)Complicating this was the fact that when a GP took on one refugee then it was most likely that the rest of the family would then come to see the same GP which could dramatically increase their caseload of patients with high health care needs. Related to this was a fear for one GP clinic of being inundated with refugees if their clinic was promoted as a formalised referral centre for refugees because of difficulties already meeting the needs of their current patient load. A situation where there was no system in place to manage referrals was described by one GP as 'a recipe for burnout'.Not having a formal referral pathway to GPs also led to problems with the transfer of health information including results from pre-departure health checks and any health services refugees had accessed in Australia. It was also noted that poor transfer of health information could also result in duplication of services such as immunisation.RemunerationHalf of the GPs identified remuneration as a challenge when working with refugees. This was because refugees were mostly bulk billed and many needed longer consultations which were felt to be inadequately remunerated under the current Medicare billing system:...it's just not financially viable because, as we know, long complex consultations are not a way which assists you to run your practice in a way that is financially viable... (Dr 11)Remuneration was also a challenge because of the fact that refugees often missed appointments, which meant no remuneration, and work with refugees often involved time consuming extra-consultation activities that could not be charged to Medicare:Missed appointments are fairly common...So you miss the remuneration if they don't come. The time factor; the complexity of the consult is more than what is remunerated for the time involved, because your follow-up is often phone calls to various agencies or organising things, writing letters, becoming an advocate, coordinating allied health. Very little of that is remunerated... (Dr 10)Despite the strong indication that remuneration was a challenge for GPs, the majority of GPs said that they were either not going to use or were unlikely to use the new Medicare item number for performing an initial health assessment on a refugee although there was moderate support for it in principle. This was either because of a lack of familiarity with already existing Enhanced Primary Care (EPC) item numbers or because of the high administrative burden associated with their use:It is not something I am likely to use personally. I think it is a great idea ... but I am not very good at using the specific numbers ... and the EPC items and so on. I just don't have time to sort out all the paperwork for that sort of thing. (Dr 6)Another GP was disappointed that the Divisions of General Practice had not produced a template to assist using the item number in the same way that they had with previous EPC item numbers. Finally, a number of GPs were critical that the item number did not go far enough in that it failed to recognise that the greater health care needs of refugees were ongoing.Infrastructure supports to perform initial assessmentsThere was strong evidence provided by the majority of GPs in this study that they did not have the necessary infrastructure support, i.e. the systems and support staff, to perform initial refugee health assessments:We are not well enough equipped. We are not resourced, we do not have the supporting background structure. (Dr 9)It was particularly overwhelming for GPs when groups or families all came at once and they had not had a previous health assessment:...we were having whole families of recent arrivals come to the surgery and need all their history taken; immunisations brought up-to-date and that was just overwhelming... We do have a practice nurse, but she is usually quite busy doing other things. That was too much for us to handle. (Dr 6)The Divisions also believed that a lack of infrastructure support was a reason it would be difficult for GPs to perform initial health assessments and was a reason uptake of the new item number would be limited:If you just basically say \"Here is a new item number doctors\" it won't be taken up, because it is going to be all too hard. From an infrastructure perspective most practices lack the infrastructure to really make this work. (Div 3)Challenges for Divisions assisting GPsThe Divisions identified a number of challenges in assisting GPs to provide care to refugees. They expressed concerns that they did not know how many refugees were being resettled as well as precisely where these resettlements were occurring within their Divisions. The Divisions reported also having limited awareness of which GPs in their Divisions were providing care to refugees and, as a result, were not able to determine which GPs might need extra support to do this work. One Division had surveyed GPs to assess this but most had been too busy to respond. Although a number of GPs had been recruited by the IHSS provider to offer care to newly arrived refugees, the Divisions expressed frustration that they did not know who these GPs were. They were also concerned that there was no collaboration with the IHSS provider which might avoid resettling refugees in areas where they might have difficulty accessing GP services:... there is no point putting refugees in to an area where there are no GPs. There might be GPs there but they might not want to see refugees or they might have closed their books. We would have that intelligence; they would have no idea about that... (Div 1)All Divisions mentioned a lack of funding as a major reason their ability to help GPs was limited. Because refugee health was not a priority area for the Commonwealth, Divisions received no direct funding for refugee health initiatives:We are funded by the Department of Health and Ageing; we have got a whole lot of quality indicators that we have got to actually achieve in. Refugee health does not even appear in there. (Div 3)The Divisions explained that it was also a lack of funding that limited their ability to assist GPs to utilise the new item number. Despite this, two Divisions had diverted core funding to better support GPs in the area of refugee health but no Divisions had funding for specific services or programs. One Division felt their case to argue for increased funding was limited given the lack of data related to refugee numbers settling in the Division and that there was no empirical evidence that GPs weren't coping even though it was recognised that GPs were too busy to answer surveys that might provide this data.Ways GPs could be better supportedProviding GPs with more resourcesDespite GPs questioning how realistic it was for them to manage many of the exotic health conditions in newly refugees, there was some support for the provision of screening guidelines for use by GPs in private practice provided they were in a simple and readily accessible format – such as linked to the general practice software program Medical Director (MD).GPs reported they could be assisted to overcome some of the challenges related to culture if they were provided with more background information about refugee groups either through the provision of information sheets or talks from different community members regarding different cultural practices. It was also suggested that these challenges could be reduced through better provision by settlement services of health information to refugees on arrival including information about common conditions in refugees such as hepatitis B, early intervention and illness prevention activities and better initial orientation to the Australian health care system. Many GPs also believed that settlement services could provide more assistance to refugees to attend appointments. Other ways of improving refugee navigation of the health system included a greater role for voluntary organisations, practice nurses or community health care workers who could be health advocates for refugees:At times you feel like you're running around doing a lot of the work that could be partly done through either a practice nurse or another allied health worker or somebody who can, on the ground, advocate for that refugee individual. (Dr 10)In relation to referral pathways of refugees to general practice, it was strongly stated by a number of GPs that this should involve a consent process which assessed the ability of a GP to take on the care of a new refugee or refugee family:It is not a kind of fair system to plonk someone onto a practice. I think agencies should actually liaise with either one of the senior doctors or, if there is a nurse... so a referral can actually be properly organised and assessed as to whether the practice can take someone on. I think the ways it has happened in the past have been really unsatisfactory. (Dr 11)Despite limited enthusiasm for the new Medicare item number, a small number of GPs were still interested in learning more about how to use it through their Division and there was also some support for the provision of a template by the Divisions which could also be incorporated into MD.The Divisions suggested a number of ways they could assist GPs to perform initial assessments if they were provided with funding. This included improving the utilisation of the item number by educating practice nurses and developing a template as well as Division funded 'clinical attachments' at the state funded specialist refugee service to provide GPs with greater expertise in managing health conditions unique to refuges. It was also suggested that Division funded refugee health infrastructure grants could provide assistance to GPs to better set up their clinics to care for refugees via IT support and the provision of business cases for employing practice nurses. One Division, however, expressed uncertainty about the likelihood that GPs would be willing to build such systems into their practices if they were only seeing small numbers of refugees, particularly given that there was limited enthusiasm to build similar systems for high prevalence chronic diseases in the general population.Providing initial refugee health care via a specialist serviceDespite indicating ways they could be assisted, a number of GPs believed that the responsibility of providing initial care to refugees should not lie with GPs in private practice:There should be a front line somewhere. I don't think general practice should be the front line. (Dr 8)Instead, a system where refugees received an initial assessment via a specialist refugee or community health service was strongly supported by both GPs and the Divisions. One GP described the advantages of a community health service where a range of services could be provided to refugees in one location:I think the community health service is the best stop for an initial assessment because of the complexity of the presentations, usually, and the need for accessing a lot of different services that is really beyond most private centre GPs to be able to do that adequately. If you have got access within one building, for example, to workers who can do some of the chasing up, some of the phoning and some of the coordination, then you are much more likely to give people a good service. (Dr 10)A number of GPs indicated that they would be much happier to accept referrals of refugees if they had had an initial assessment where they could then focus on their more day-to-day health needs.DiscussionThis study highlights the many and diverse challenges faced by GPs in private practice when providing health care to refugees in their initial resettlement period. These challenges and their policy implications are discussed below.Challenges performing initial assessmentsThe extent of the challenges faced by GPs providing initial care to refugees in this study is not surprising given that it is during their early resettlement period that refugees are most likely to experience multiple medical problems, many of an exotic nature, when language and cultural barriers are likely to be greatest and when refugee knowledge of the Australian health care system and general health literacy are likely to be most limited.Whilst many of the challenges identified support previous research in this area, the most striking feature of this study is the strong evidence that GPs in private practice are not sufficiently resourced to provide initial care effectively to newly arrived refugees with multiple and complex health needs. For GPs in this study, the lack of resources existed both at an individual GP level, with GPs lacking comprehensive knowledge of the health conditions unique to refugees, as well as at a more structural health system level, where GPs lacked both the time and the infrastructure support to do this work effectively. Further, the lack of resources was not related to the length of time GPs had been providing care to refugees or the intensity of their involvement other than that GPs with less experience were less familiar with the use of interpreter services.Given that the first point of contact with the Australian healthcare system for refugees currently arriving in SA is with a GP in private practice, there are a number of health consequences for refugees if GPs do not have the necessary resources to provide them with effective care in their initial resettlement period. These include GPs missing or inadequately treating physical and mental health problems unique to refugees, refugees not following through with treatments or referrals and refugees under engaging with illness prevention activities because of poor health literacy [30]. This can result in refugees experiencing a reduced health status compared with the non-refugee population as well as potentially greater costs to the health system because of later and more expensive treatments.Implications for policyBarriers to building capacity of GPs to perform initial assessmentsAlthough GPs mentioned a number of ways they could be assisted to provide more effective initial care to refugees, they also indicated that there would be major limitations attempting to build this capacity.Firstly, GPs questioned the expectation that they develop the specific 'refugee health' expertise needed for performing initial assessments which competed with their role as 'generalists'. It is likely that, at best, developing the required expertise to perform initial assessments will only appeal to a small number of GPs.Secondly, even those GPs who have an interest in doing this work may not want to identify themselves as a 'refugee doctor' for fears, as stated by one GP, that they will become inundated with referrals. GPs in this study indicated that there were limits to the amount of work they could do with refugee patients given the often multiple and complex needs on initial presentation. GPs, however, operate in a primary health care (PHC) system where they have little control over how patients are referred to them. Further, GPs performing initial health assessments are most likely to be the GPs who provide the ongoing care (more so now given that refugees are initially settled in more permanent accommodation). As a result, GPs providing initial health care can quickly end up with very high numbers of patients with multiple and complex health needs. A number of GPs in this study indicated that this had contributed to them closing their books to new patients. Refugee health service providers in Adelaide as well as interstate[2] have also experienced the difficulties sustaining GP involvement under these circumstances. To avoid overburdening a small number of GPs would mean, however, offering more general training to a large number of GPs. This is unlikely to be a cost effective approach and also, as evidenced by this study, developing the necessary expertise and building the practice systems required to provide effective initial health care to refugees will, at best, appeal only to a small number of GPs.Thirdly, GPs indicated that providing initial care to refugees was time consuming but the fee-for-service structure of general practice combined with GP workforce shortages limited the time GPs could offer to refugees to manage their multiple and complex health needs – a problem shared with other groups who have greater health care needs [31]. Under these circumstances, GPs are unlikely to take on a role that will require them to offer a greater number of longer consultations. This could be one reason why the new Medicare item number received limited support from GPs in this study despite the fact that a lack of remuneration was an issue for a number of them. As suggested by the Divisions, this could also be an indication that the new item number, in its current form, does very little to address the resource problems described above. It is interesting to note that the initial uptake of the item number was greatest in Victoria [32] where a large number of refugees receive initial GP care in community health centres with the support of refugee health nurses which highlights the importance of providing adequate time and infrastructure support when doing this work [33]. A further limitation of the item number, also mentioned by GPs in this study, is that it does not take into account the fact that the greater initial health care needs often persist beyond the first visit with a GP.The role of a specialist health serviceAn alternative to providing initial care to refugees in private general practice is for this to be provided in a specialist refugee service or community health setting. Such a service delivery model received strong support from participants in this study. Previous studies have similarly highlighted the central importance of community health services providing initial health care to patients with complex health needs, including refugees, as a result of better access to resources and infrastructure support [2,26,34].It is acknowledged that there is not a one size fits all approach when determining which model, specialist or mainstream, best meets the special service needs of refugees in their initial resettlement period [35] and that receiving PHC via a specialist service may delay refugee engagement with local mainstream PHC services [2]. Where there is limited capacity, for mainstream services to provide for these special needs, and this study provides strong evidence that this is the case in private general practice in SA, there is a role for a specialist service to fill this service gap [35,36]. In SA, such a state funded service already exists, although it is not currently being utilised as the current settlement service provider has adopted a policy of connecting refugees directly with mainstream health services immediately on arrival to Adelaide. It makes sense to utilise the current refugee health expertise and resources of this state funded service to provide initial health care services to refugees, especially those with complex health needs and significant resettlement challenges. Whilst this is a centralised service, the highly centralised population distribution of SA in Adelaide, combined with the relatively small number of refugee arrivals, means that it is accessible for the majority of refugees in SA. Further, the ability to deliver refugee services in multiple community health locations, such as in Victoria, is limited in Adelaide because of a lack of medical presence at these sites. It is recognised, however, that a centralised specialist service is not well suited to larger Australian cities such as Sydney and Melbourne. Finally, if initial health assessments are provided by a specialist service, it is important that a clear, transparent and effective referral system to a nominated general practice is part of this process when initial health care needs have been met. Ongoing links between general practices and the specialist provider would also address a number of the other challenges identified by GPs and Divisions in this research.Study strengths and limitationsThis study provided a unique and detailed insight into the experience of GPs providing health care to refugees. However, given the small number of participants in this study, these results cannot be generalised to all GPs in Adelaide or GPs in other locations. To do this, a larger quantitative study would be required. The low response rate from GPs could have meant that those GPs involved were more motivated to participate because of dissatisfaction with the current system of provision of initial health care to refugees. This low response rate, however, mirrors the experience of Divisions in this study and their difficulties getting GPs to participate in research and respond to surveys. Further, it is generally believed that at the time data was collected for this study there were a limited number of GPs (although the exact number is not available) providing initial care to refugees in SA. It is likely, therefore, that the views of GPs in this study not only provide a reasonably comprehensive summary of the challenges of providing initial care but are also the experience of most GPs doing this work in SA at the time. Whilst GPs interstate are likely to face many similar challenges providing initial care to refugees, it is beyond the scope of this paper to comment on how well resourced they are to provide effective care.ConclusionThis study provides evidence that, due to a range of challenges, GPs in private practice in SA are insufficiently resourced to provide initial health care effectively to refugees and that attempting to overcome these challenges would face a number of obstacles. Whilst further evidence is required to document the extent of these challenges in SA and how they might be best addressed, it makes sense for the existing state funded refugee health service to be involved in the delivery of initial PHC services to refugees, especially those with complex health needs and significant resettlement challenges.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsDJ conceptualised the study, conducted the literature review, undertook data collection and analysis, and drafted the paper. AZ and TB advised on all stages of the work including analysis of the data as well as reviewing and contributing to drafts of the paper. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2531180",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531180\nAUTHORS: Tonino Bombardini, Vincenzo Gemignani, Elisabetta Bianchini, Lucia Venneri, Christina Petersen, Emilio Pasanisi, Lorenza Pratali, Mascia Pianelli, Francesco Faita, Massimo Giannoni, Giorgio Arpesella, Eugenio Picano\n\nABSTRACT:\nBackgroundRecently, a cutaneous force-frequency relation recording system based on first heart sound amplitude vibrations has been validated. A further application is the assessment of Second Heart Sound (S2) amplitude variations at increasing heart rates. The aim of this study was to assess the relationship between second heart sound amplitude variations at increasing heart rates and hemodynamic changes.MethodsThe transcutaneous force sensor was positioned in the precordial region in 146 consecutive patients referred for exercise (n = 99), dipyridamole (n = 41), or pacing stress (n = 6). The curve of S2 peak amplitude variation as a function of heart rate was computed as the increment with respect to the resting value.ResultsA consistent S2 signal was obtained in all patients. Baseline S2 was 7.2 ± 3.3 mg, increasing to 12.7 ± 7.7 mg at peak stress. S2 percentage increase was + 133 ± 104% in the 99 exercise, + 2 ± 22% in the 41 dipyridamole, and + 31 ± 27% in the 6 pacing patients (p < 0.05). Significant determinants of S2 amplitude were blood pressure, heart rate, and cardiac index with best correlation (R = .57) for mean pressure.ConclusionS2 recording quantitatively documents systemic pressure changes.\n\nBODY:\nIntroductionRecently, a cutaneous operator independent force-frequency relation recording system as been validated in the stress echo lab, based on first heart sound amplitude variations at increasing heart rates [1,2]. Contractility quantification and systolic/diastolic time measurement through the system has been previously demonstrated [1,3]. A further application could be the assessment of Second Heart Sound (S2) amplitude variations at increasing heart rates. In fact, the maximum amplitude of vibrations measured by the sensor following the ECG T wave originates from the physical phenomenon of the abrupt deceleration of the moving aortic blood mass. The audible components of this deceleration give rise to the Second Heart Sound (S2) [4-6]. The aim of this study was to assess the relationship between second heart sound amplitude variations at increasing heart rates and hemodynamic changes.MethodsPatient selectionWe enrolled 146 consecutive patients (99 males, 60 ± 14 years) referred for stress echocardiography. Patients' characteristics are summarized in Table 1. The type of stressor was chosen by the attending cardiologist/echocardiographist at time of testing in relation to relative contraindications of one stressor over the other [7,8]. Ninety-nine subjects underwent exercise stress (13 non competitive athletes were the controls). Twenty-four patients unable to exercise and 17 patients scheduled for coronary flow reserve evaluation underwent dipyridamole stress echo. Six patients with permanent pace maker (DDD in 5, BIV in 1) underwent pacing stress. Coronary artery disease was defined by the presence of angiographically assessed coronary stenosis (with quantitatively assessed diameter reduction in major coronary vessels) or previous myocardial infarction. The local Ethical Committee approved the study protocol. All patients gave their written informed consent before entering the study. All patients met the following inclusion criteria: 1) referred to stress echo for clinically-driven testing. 2) acoustic window of acceptable quality 3) willingness to enter the study. From the initially considered population of 152 patients, 4 were excluded for poor acoustic window (n = 4), or refusal to give written informed consent (n = 2).Table 1Characteristics of the study patientsEXERCISEDIPPACINGPt n°99416Age (years)56 ± 1468 ± 1168 ± 10Males68274Controls13--CAD36293Previous PTCA/By pass27191Previous myocardial infarction25132Arterial hypertension185-Valvular disease1921Atipical chest pain1231DCM121Semi-supine bicycle exerciseGraded bicycle semi-supine exercise echo was performed starting at an initial workload of 25 watts lasting for 2 minutes; thereafter the workload was increased stepwise by 25 watts at 2 minutes interval. A 12-lead electrocardiogram and blood pressure determination were performed at baseline and every minute thereafter [7]. Two-dimensional echocardiographic monitoring was performed throughout and up to 5 min after the end of peak stress. Two-dimensional images were recorded at baseline and at the end of each step.Dipyridamole stress echoTwo-dimensional echocardiography and 12-lead electrocardiographic (ECG) monitoring were performed in combination with high dose dipyridamole (up to 0.84 mg over 6 min) in accordance to well established protocols [7,8]. Contraindications to using dipyridamole were asthma, hypotension, and bradyarrhythmias.Pacing stress echoThe pacing protocol was accelerated (with a 10-beat increment every 60 s) until one of the following criteria was reached: 1 – 85% of maximal heart rate (age-corrected: 220 – age for men, 200 – age for women); or 2 – PM maximal programmable heart rate (which varied widely, according to the model of PM, up to 170 bpm during stress). Stimulation was performed, wherever possible, in atrial stimulation mode, or dual-chamber (DDD) pacing to have normal contraction sequence [9].Regional wall motion analysisRegional wall motion analysis was evaluated at baseline and at peak stress with a semiquantitative assessment of a wall motion score index (WMSI), with the 17 segment model of the left ventricle, each segment ranging from 1 = normal/hyperkinetic to 4 = dyskinetic, according to the recommendations of the American Heart Association and American Society of Echocardiography. WMSI was derived by dividing the sum of individual segment scores by the number of interpretable segments [8,10]. Test positivity was defined as the occurrence of at least one of the following conditions: 1) new dyssynergy in a region with normal rest function (i.e., normokinesia becoming hypokinesia, akinesia or dyskinesia) in at least two adjacent segments.Diagnostic end points and interruption criteriaThe diagnostic end-points for all types of stress were: the development of obvious echocardiography positivity. Non-echocardiographic test end-points were the following: peak dipyridamole dose; 85% of target heart rate; achievement of conventional end-points (such as severe chest pain and/or diagnostic ST segment changes). The test was also stopped, in the absence of diagnostic endpoints, for one of the following reasons of constituting a submaximal, non-diagnostic test: intolerable symptoms; limiting asymptomatic side effects, consisting of: a) hypertension (systolic blood pressure >220 mmHg; diastolic blood pressure >120 mmHg); b) hypotension (relative or absolute): >30 mmHg fall of blood pressure; c) supraventricular arrhythmias: supraventricular tachycardia or atrial fibrillation; d) ventricular arrhythmias: ventricular tachycardia; frequent, polymorphous premature ventricular beats [8].Blood pressure analysisOne nurse recorded blood pressures at rest and during each individual study. The blood pressure recording was made using a sphygmomanometer and the diaphragm of a standard stethoscope. Systolic and diastolic blood pressure was obtained in the right arm. During exercise test, blood pressure recording was obtained with patient lying in a left rotated semi supine position and instructed to hand grip to the left support with their left hand. Patients have been told to let their right hand go limp when blood pressure was measured.By selection, 75 out of the 99 patients of the exercise group had simultaneous S2 amplitude and systemic blood pressure measurement at the first, third and fifth post exercise minute time.Volume analysisAll patients underwent transthoracic echocardiography at baseline and during stress. Left ventricular end-diastolic and end-systolic volumes were measured from apical four- and two-chamber view, by an experienced observer using the biplane Simpson-method. Only representative cycles with optimal endocardial visualization were measured and the average of three measurements was taken. The endocardial border was traced, excluding the papillary muscles. The frame captured at the R wave of the ECG was considered to be the end-diastolic frame, and the frame with the smallest left ventricular cavity the end systolic frame. Images were acquired at baseline and at each increase in heart rate of 10 beats during stress.Systemic Vascular Resistance (SVR)SVR were calculated according to the traditional formula:SVR = 80 * (MAP-5)/CO,where 5 is an approximation of the right atrial pressure and MAP is mean arterial pressure.Systemic arterial complianceSystemic arterial compliance (C) was calculated as stroke volume index/systemic arterial pulse pressure; were pulse pressure = systolic blood pressure – diastolic blood pressure [11].Arterial elastance and ventricular-arterial couplingIn all, ventricular arterial coupling was indexed by the ratio of left ventricular systolic elastance index (systolic pressure/end-systolic volume index) to arterial elastance (Ea, ratio of end-systolic pressure by stroke volume). Echocardiography (for ESV and stroke volume) and cuff sphygmomanometer (systolic pressure, multiplied × 0.90 to obtain end-systolic pressure) provided the raw measurements.Because stroke volume (and input impedance) varies directly with body size, arterial elastance was adjusted for body surface area (EaI) to better reflect differences in arterial properties with age and between the genders adjusted for differences in body size [12]. Of note ventricular-arterial coupling is ventricular elastance/arterial elastance, which can further be described as: end-systolic pressure/end-systolic LV volume divided by end-systolic pressure/stroke volume: the pressure terms in the numerator and the denominator cancel out, and ventricular-arterial coupling equals to stroke volume/end-systolic volume.Operator-independent second heart sound quantificationThe transcutaneous force sensor is based on a linear accelerometer from STMicroelectronics (LIS3). The device includes in one single package a MEMS sensor that measures a capacitance variation in response to movement or inclination and a factory trimmed interface chip that converts the capacitance variations into analog signal proportional to the motion. The device has a full scale of ± 2·g (g = 9.8 m/s2) with a resolution of 0.0005·g. We housed the device in a small case (Figure 1) which was positioned in the mid-sternal precordial region and was fastened by a solid gel ECG electrode. The acceleration signal was converted to digital and recorded by a laptop PC, together with an ECG signal. The system is also provided with a user interface that shows both the acceleration and the ECG signals while the acquisition is in progress[1]. The data were analyzed by using software developed in Matlab (The MathWorks, Inc). A peak detection algorithm, synchronized with the ECG, scans the first 150 ms following the R wave to locate the first heart sound vibration. Subsequently, the interval between the first heart sound and the following R wave is analyzed to record the amplitude (nadir to peak) of second heart sound vibration for each cardiac beat [3]. The accelerometer simply records naturally generated heart vibrations, which audible components give rise to the second heart sound 'See additional file 1: Appendix'.Figure 1Isovolumic contraction force and second heart sound (S2) amplitude. A Micro-Electro-Mechanical Systems (MEMS) accelerometer is temporarily positioned in the mid-sternal precordial region before starting the scheduled stress test in all patients. A peak detection algorithm, synchronized with the ECG, scans the first 150 ms following the R wave to record the isovolumic contraction force vibration and then the interval before the following R wave to record the second heart sound amplitude (S2, pink symbol). All the parameters are acquired as instantaneous values at baseline and during stress. The data can be also read remotely by a wireless bluetooth sensor network, with reliable continuous remote monitoring 'See additional file 1: Appendix'.The curve of S2 peak amplitude variation as a function of heart rate was finally computed as the increment with respect to the resting amplitude value [13]. All the parameters were acquired as instantaneous values at baseline and during stress; mobile mean was utilized to assess baseline value (1 minute recording), at each incremental stress test, at peak test, and during recovery (Figure 2). Baseline, peak stress, peak-rest difference as absolute value, and delta % rest-peak stress values were computed.Figure 2Computing the second heart sound amplitude variation as a function of heart rate. All the parameters are acquired as instantaneous values at baseline and during stress; mobile mean is utilized to assess baseline value (1 minute recording), at each incremental stress test, at peak test, and during recovery. Left panel: instantaneous S2 amplitude scattering (blue points exercise, red points recovery) depends on the respiratory cycle and thorax expansion; blue and red curves = S2 amplitude mobile mean. Right panel: blue curve = exercise in progress; red curve = recovery.Non myocardial noising vibrations (skeletal muscles, body movements, breathing) were eliminated by frequency filtering.Statistical analysisSPSS 11 for Windows was utilized for statistical analysis. The statistical analyses included descriptive statistics (frequency and percentage of categorical variables and mean and standard deviation of continuous variables).The one-way ANOVA was used to compare continuous variables between groups; when homogeneity of variance was not present, the Kruskal-Wallis test for nonparametric independent samples was used. Intergroup comparison was performed with Scheffe and Tamhane post hoc tests, respectively.Relations between variables were assessed using linear regression analysis and Pearson's correlation coefficient. Cardiac or vascular properties significantly related to the S2 amplitude changes were graphically displayed with simple scatter plots. Crosstabs' statistics and measures of association for post exercise hypotension vs. post exercise S2 amplitude undershoot were performed in 75 selected patients.ResultsResting and stress echocardiographic findingsTechnically adequate images were obtained in all patients at baseline (by selection) and during stress.At Peak ExerciseHeart rate was lower in the dipyridamole than in the exercise and pacing groups. The mean ejection fraction increased in the exercise and Dip groups, while decreased in the pacing group. Regional wall motion abnormalities occurred in 5 patients of the exercise, 1 patient of Dip and 2 patients of the pacing groups (Table 2).Table 2Rest and stress dataEXERCISEDIPPACINGN of pts99416Age (yrs)56 ± 14§68 ± 1168 ± 10Gender (M/F)68/3127/144/2BSA (m2)1.88 ± .191.83 ± .161.87 ± .28Standard echo measurementsLVMI (g/m2)104 ± 28104 ± 20138 ± 34HR rest (bpm)73 ± 1666 ± 1371 ± 10HR peak (bpm)131 ± 24Δ84 ± 13*132 ± 13LV EF % rest59 ± 1158 ± 1351 ± 11LV EF % peak67 ± 14‡62 ± 13*45 ± 16WMSI rest1.11 ± .291.17 ± .321.28 ± .46WMSI peak1.13 ± .311.19 ± .321.4 ± .46Δ WMSI (rest-peak).02 ± .10.01 ± .07.15 ± .24Sensor built second heart sound (S2) amplitude changesS2 rest (mg)7.7 ± 4.97.1 ± 2.85.8 ± 1.4S2 peak (mg)15.9 ± 8.7§7.2 ± 37.7 ± 2.4S2 Δ rest-peak (mg)8.2 ± 6.1§.1 ± 1.51.8 ± 1.9S2 Δ % (rest-peak)133 ± 104§2 ± 2231 ± 27Perpheral pressures, load and couplingSBP rest (mmHg)134 ± 21137 ± 20131 ± 25SBP peak (mmHg)189 ± 26§127 ± 26137 ± 37Δ SBP (rest-peak, mmHg)55 ± 25§-8 ± 176 ± 17DBP rest (mmHg)74 ± 1271 ± 1274 ± 11DBP peak (mmHg)94 ± 13§67 ± 1375 ± 15Δ DBP (rest-peak, mmHg)20 ± 13§-4 ± 101 ± 15Mean pressure rest (mmHg)94 ± 1393 ± 1293 ± 14Mean pressure peak (mmHg)126 ± 15§88 ± 1796 ± 20Δ mean pressure (rest-peak, mmHg)32 ± 14§-5 ± 122 ± 15SVR rest (dyne * sec * cm-5)2134 ± 8022118 ± 7021652 ± 533SVR peak (dyne * sec * cm-5)1501 ± 5471551 ± 7471546 ± 620Δ SVR (rest-peak, dyne * sec * cm-5)-632 ± 669‡-567 ± 613-106 ± 382Arterial compliance rest (mL *m-2/mmHg)0.49 ± 0.180.48 ± 0.20.7 ± 0.38Arterial compliance peak (mL *m-2/mmHg)0.33 ± 0.11Δ0.55 ± 0.220.4 ± 0.2Δ Arterial compliance (rest-peak, mL *m-2/mmHg)-0.17 ± 0.17Δ0.07 ± 0.15*-0.3 ± 0.24Arterial elastance index rest (mmHg/mL/m2)4.7 ± 1.54.5 ± 1.53.6 ± 1.1Arterial elastance index peak (mmHg/mL/m2)6.2 ± 1.8Δ4.1 ± 1.1*6.3 ± 2.5Δ Arterial elastance index (rest-peak, mmHg/mL/m2)1.5 ± 1.6Δ-.4 ± 1.4*2.7 ± 1.7Ventricular/arterial coupling rest (SP/ESV/EaI ratio)1.8 ± .91.8 ± .91.3 ± .7Ventricular/arterial coupling peak (SP/ESV/EaI ratio)2.9 ± 1.9§2.1 ± 1.11.1 ± .8Δ Ventricular/arterial coupling (rest-peak)1.1 ± 1.6§.4 ± .6-0.2 ± .4Cardiac index rest (L/min/m2)2 ± 0.71.9 ± 0.52.5 ± 0.7Cardiac index peak (L/min/m2)3.9 ± 1.3§2.7 ± 0.92.7 ± 0.8Δ Cardiac index (rest-peak, L/min/m2)1.9 ± 1.2§0.7 ± 0.60.3 ± 0.5§= significant differences between exercise and both dipyridamole and pacing stress pts; ‡ = significant differences between exercise and pacing stress pts; * = significant differences between dipyridamole and pacing stress pts; Δ = significant differences between exercise and dipyridamole stress ptsPeripheral pressures, load and couplingArterial elastance increased in the exercise and the Pacing groups, while decreased in the dipyridamole, mainly due to a greater dipyridamole induced arterial compliance (Table 2).Despite similar baseline values, diastolic blood pressure increased in the exercise, decreased in the dipyridamole, while unchanged in the pacing group, although the response was heterogeneous at the individual level (Table 2).Sensor built second heart sound amplitude variationsA consistent second heart sound signal was obtained in all patients at rest and during stress (Figure 2). In the patients as a whole, baseline S2 was 7.2 ± 3.3 mg, increasing to 12.7 ± 7.7 mg at peak stress. S2 trends during exercise or dipyridamole are shown in Figure 3.Figure 3Second heart sound (S2) amplitude recording simultaneously with diastolic blood pressure during stress. Left panel: similar S2-frequency trend during stress (blue symbols) and recovery (red symbols) in a patient with normal exercise pressure changes and without post exercise hypotension. Middle panel: S2-frequency trend during stress (blue symbols) and recovery (red symbols) in a patient with exercise induced diastolic hypertension and post exercise hypotension. Right panel: flat-negative S2-frequency trend during dipyridamole stress induced hypotension.Mean S2 percentage increase was + 133 ± 104% in the 99 exercise patients, + 2 ± 22% in the 41 dipyridamole patients and + 31 ± 27% in the 6 pacing patients (p < 0.05 between groups) (Table 2).In the exercise group the S2 amplitude percentage increase was similar in the 13 control and in the 86 patients (+ 140 ± 123% vs. 132 ± 102%, p = ns)At linear regression analysis significant positive determinants of the S2 amplitude changes during stress were the systemic blood pressure, the heart rate, and cardiac index rest-peak changes (Table 3). Scatter plots demonstrating correlations between S2 changes and arterial pressure rest-peak changes are displayed in Figure 4.Table 3Significant determinants of the sensor second heart sound (S2) amplitude valuesRest S2Peak S2S2 Δ % rest-peakAge (yrs)-.359 (<.01)-.476 (<.01)-.153 (<.05)BSA (m2)LVMI (g/m2)-.194 (<.05)LV EF %.215 (<.01)WMSIHR (bpm).206 (<.01).516 (<.01).453 (<.01)Diastolic Blood Pressure (mmHg).183 (<.05).319 (<.01).502 (<.01)Systolic Blood Pressure (mmHg).338 (<.01).544 (<.01)Mean Blood Pressure (mmHg).345 (<.01).567 (<.01)Ventricular elastance (mmHg/mL/m2).144 (<.05).218 (<.01)Arterial elastance.307 (<.01).281 (<.01)SVR (dyne * sec * cm-5)Arterial compliance (mL *m-2/mmHg)-.340 (<.01)-.300 (<.01)Ventricular/arterial couplingCardiac index.153 (<.05).432 (<.01).388 (<.01)Linear regression analysis to identify significant relationship between predictor variables (first column) and the sensor second heart sound (S2) amplitude was performed for baseline (second column) peak stress (third column) and rest-peak delta values (fourth column).Pearson's correlation coefficients (and significance value within brackets) are reported in cells with significant (p < 0.05) relationships.Figure 4Second Heart sound recording quantitatively documents systemic pressure changes. Scatter plots demonstrating relationship between sensor Second Heart Sound amplitude % changes (y axis) and systemic pressure rest-peak changes values (x axis) in the whole group of 146 patients. Red symbols: exercise stress; green symbols: dipyridamole stress; blue symbols: pacing stress. Left panel: systolic pressure (SBP) changes. Middle panel: diastolic pressure (DBP) changes. Right panel: mean pressure (MP) changes.Second heart sound undershoot and the post exercise hypotensionA significant correlation was found between post exercise hypotension and recovery S2 undershoot: 44 (80%) of the 55 patients with post-exercise hypotension had S2 undershoot in the recovery, while 19 (96%) of the 20 patients without post-exercise hypotension had stable rate-S2 curve at recovery (Table 4) (Figure 3).Table 4Crosstabs' statistics and measures of association for post exercise hypotension vs. post exercise second heart sound amplitude undershoot in 75 selected patientsExercise recovery hypotensionExercise recovery isopressureTotalSHS Recovery Under shot44145SHS Recovery same shot111930Total552075Kendall's tau-c = 0.591 P < 0.001DiscussionA stable, reproducible, and consistent S2 force signal was recorded in all patients at rest and during stress. Baseline force value had an ample range (from 2 to 23 g * 10-3). The most widely accepted theory for the genesis of the second heart sound is the \"cardiohemic model,\" which states that the sounds are produced by the vibration of the entire heart and its contents [14]. This vibration is triggered by valve closure (the aortic and pulmonary valves for the second heart sound). The amplitude of these sounds depends on the force with which the valves close, which, in turn, depends on the pressure gradient across the valve at the time of closure. We previously demonstrated that in adult patients undergoing stress testing, the first heart sound amplitude was directly related to myocardial contractility [1]. In this investigation, blood pressure (systolic, diastolic and mean) correlated closely with S2 amplitude. This may be explained by the fact that amplitude is primarily determined by one factor, the force of valve closure [15].Biophysics of the second heart soundEarly studies of the hemodynamic determinants of the amplitude of the S2 have related the aortic component amplitude of the S2 vibration to the aortic pressure, in agreement with clinical findings that hypertensive patients frequently have loud second heart sounds [4,16,17]. In their proposed mechanism for the origin of the second heart sound, Sabbah and Stein [6] showed a relation between the amplitude of S2 and the driving pressure. Driving pressure, in the heart, refers to the instantaneous difference between arterial and ventricular pressure shortly after semilunar closure. Kusukawa and associates [5] previously found a good correlation of the amplitude of the second heart sound with the difference of pressure between the aorta and the left ventricle coincident with the dicrotic notch. But patients suffering from myocardial infarction and/or heart failure, often exhibit reduced S2 amplitude, even when the aortic pressure is normal [18]. They showed that the amplitude of S2 was linearly related to the rate of change of the pressure gradient that develops across the aortic valve during diastole (r = .82). The latter is also correlated with negative dP/dt (r = .62).In normotensive patients with poor ventricular performance, the rate of isovolumic relaxation may be compromised and this would cause a reduction in negative dP/dt which in turn causes a reduction of the rate of change of the pressure gradient that develops across the valve during diastole. A diminished S2, therefore, would result due to the more slowly developing driving pressure, which directly affects the characteristics of valvular vibration. Tanigawa et al [4] demonstrated in instrumented dogs, that when the time constant of left ventricular pressure fall \"T\" was normal, the aortic systolic pressure and diastolic pressure were good predictors of S2 intensity. When LV relaxation was impaired, increasing T greater than 135% of control, the S2 intensity for any given aortic pressure was reduced. When relaxation was hyperactive, decreasing T less than 65% of control, S2 intensity was increased. Aortic pressure/T which assessed both aortic pressure and relaxation ability, is a better determinant of A2 intensity than aortic systolic pressure or aortic diastolic pressure alone.Second heart sound frequencies or amplitude to get clinical information?Previous studies have shown that it is possible to estimate systemic blood pressure using the spectral information of the second heart sound. A mathematical model for the vibration of the closed aortic valve was proposed by Zhang et al [19], showing that the increasing aortic pressure results in an increase both in frequency and amplitude of produced sound. The results of this study also suggest that it is the increasing resonant frequency and amplitude of the blood column induced by elevated distending pressure that plays significant role in the process.Various mathematical methods have been used to describe heart sounds, including the frequency domain (FFT) and the time domain (RMS) amplitude.The frequency domain (FFT)The frequencies present in heart sounds are determined by the volume of the vibrating mass (smaller volume has a higher resonance frequency) and the tension generated in the walls of the heart and great vessels. This explains the fact that S2 is normally of higher frequency than S1 (the aorta is of lower volume than the heart) and that younger children exhibited higher heart sound frequencies than older children [20]. Other Authors [21] stated that the major concentration of energy, for both first heart sound (M1) and second heart sound (S2), is below 150 Hertz (Hz) which may indicate that both sounds are caused by vibrations within the same structure, possibly the entire heart. However S2 spectra have greater amplitude than S1 spectra above 150 Hz, which may be due to vibrations within the aorta and pulmonary artery. Because peak frequency is a descriptor of only a single point, it is therefore not a useful factor in describing heart sound changes resulting from variations in myocardial contractility or systemic pressure changes 'See additional file 1: Appendix'.The amplitude domain (RMS)In previous investigation [20], hemodynamic variables (heart rate and blood pressure) correlated more closely with amplitude than with frequency. This may be explained by the fact that amplitude is primarily determined by one factor – force of valve closure – whereas frequency depends on the force of closure, heart volume, and the resonance frequencies of the heart and great vessels. Thus, differences in heart size and intravascular volume status could explain the greater variability (and, thus, weaker statistical correlation) in frequency than amplitude characteristics.This is the motive for we used a peak amplitude (nadir to peak) signal analysis system for both the first and the second heart sounds vibrations [1,3].The properties of the chest wall in the transmission of sound from inside the thorax to the surface of the chestThe chest wall is a low-pass filter. Cardiac vibrations propagate as mechanical shear waves, and the intervening viscoelastic thoracic tissue attenuates the higher frequencies and introduces a variable propagation delay [22,23].In contrast to the dynamics observed epicardially, Wood [24] demonstrated that heart sound frequency law was dominated by quasi-stationary and impulse-like components implying that the instantaneous power and the power spectrum contain most of the diagnostic information in heart sound.Modelling the heart/thorax acoustic system in dogs, based on the simultaneous recording of the intracardiac and thoracic phonocardiograms, Durand and co-workers [25] showed that the heart/thorax acoustic system acts like a band pass filter having a higher attenuation for A2 than for M1. Between 20 and 100 Hz, the mean attenuation of M1 is 30 dB while that of A2 is 46 dB. Above 100 Hz, the attenuation slope is -12 dB per octave for M1 and -6 dB per octave for A2. Again, the frequency domain is influenced by the heart/thorax acoustic system, and the frequency based heart sound information is jeopardized by a further variable. Using heart sound amplitude to get clinical information, the absolute force value in the single patient is certainly related to the transthoracic propagation of cardiac vibrations. In fact, when measured epicardially or on the aortic root, S2 vibrations are up to 10 times more powerful than when measured on the chest, and cannot be used as absolute value for interpatient comparison. However the amplitude (force) % changes (i.e. contractility for M1 and systemic pressure for S2) are not influenced by the heart/thorax acoustic system and the data can be used for intrapatient changes as for contractility or systemic pressure changes.Second heart sound and stress changesPrevious phonocardiography research has been focused on the determination of heart sound production at rest, but relatively little work has been done to investigate heart sounds under stress testing. Luisada et al. [26] stated that heart sound changes during stress may be more rapid and sensitive than changes in heart rate and blood pressure. Of the 146 study patients, 39 unchanged or decreased diastolic blood pressure at peak stress vs. rest (mainly dipyridamole group, 28 out of 41 pts) while 107 increased diastolic blood pressure (mainly exercise group, 91 out of 99 pts). Patients with increased pressure had + 116 ± 106% second heart sound amplitude increase vs. + 26 ± 67% in patients with unchanged or decreased diastolic blood pressure at peak stress. In our study, a mismatch between increased diastolic pressure, but blunted S2 amplitude, occurred in 7 patients out of the 107 with stress increased diastolic pressure. According to the physiological basis, in these case the blunted S2 increase should be related to a diminished driving pressure between the aorta and the left ventricle, with delayed or altered active LV relaxation. These 7 patients had coronary artery disease. Obviously, sensor measured S2 amplitude, without ventricular relaxation data, blind us to the quantification of the time constant of left ventricular pressure fall, and/or to negative LV dP/dt. However, this totally noninvasive sensor demonstrated capability to monitor beat to beat systemic pressure changes, at rest and during exercise. Further studies with simultaneous hemodynamic in humans should be done to address this issue.Second heart sound and post exercise hypotensionPost exercise hypotension has been demonstrated both in hypertensive and healthy subjects [27] . In normotensive subjects, it has been attributed to a decrease in cardiac output and/or systemic vascular resistance [28,29]. Moreover, it has been accompanied by a decrease in peripheral sympathetic activity [29,30] and an increase in cardiac sympathetic activity [31]. Other studies demonstrated that the acute post-exercise reduction in blood pressure was clinically similar following high intensity short duration exercise and moderate intensity longer duration exercise [32]. Acute exercise may serve as a non-pharmacological aid in the treatment of hypertension. S2 amplitude monitoring could be a method to assess efficacy of the acute post-exercise blood pressure reduction. In the selected patients of our study, a significant correlation was found between post exercise hypotension and recovery second heart sound lower amplitude, to confirm the capability of the sensor to mirror diastolic pressure trend.Limitations of the studyWe used intermittent auscultatory methods to determine exercise and post-exercise blood pressure. These auscultatory methods are prone to sampling error and may provide inaccurate results. Since diastolic isovolumic relaxation occurs simultaneously with the physical phenomenon (the abrupt deceleration of the moving aortic blood mass), that gives rise to the S2 amplitude, the S2 amplitude is an algebraic sum of the myocardial and of the aortic blood mass effects. Several scenarios can occur for S2 amplitude. 1 – With constant ventricular relaxation rate, S2 amplitude is directly related to the diastolic aortic pressure: 2 – With constant aortic diastolic pressure, S2 amplitude is directly related to the ventricular relaxation rate. Obviously, sensor measured S2 amplitude, without ventricular relaxation hemodynamics, cannot sense the ventricular component of the S2. Further studies in humans with simultaneous hemodynamic assessment should be done to address this issue. Another limitation of the study could arise from the fact that we didn't measure the split in the second cardiac sound [33,34]. The continuous wavelet transforms (CWTs) method is capable of detecting its two components, A2 and P2, allowing therefore the measurement of the delay between them. This delay, called the split, is very important in the diagnosis of many pathological cases, but it was not the aim of this studyCharacteristics of the population and inducible ischemiaEight (5%) of the 146 patients had stress induced ischemia. The low rate of test positivity depends on many factors. The test indication class was not always I or IIa: low appropriateness in a high volume laboratory setting mainly depends on too often repeated tests in the absence clinical changes [35]. Second, stress test was often performed in young patients with low pre-test probability of CAD (13 controls and 39 patients with atypical chest pain and/or systemic hypertension). Third, valvular heart disease patients (moderate aortic stenosis in 9, moderate mitral regurgitation in 10) were referred for Doppler stress echo. Fourth, 17 CAD patients underwent dipyridamole stress for coronary flow reserve evaluation of left anterior descending coronary artery [8].ConclusionContinuous and non-invasive monitoring of blood pressure (BP) is important to prevent hypertensive patients from stroke and heart attack. However, most of the prevalent BP devices can provide solely intermittent measurements. S2 recording quantitatively documents systemic pressure changes: S2 amplitude trend is up-sloping when pressure increases as may occur during physical exercise or is flat for a flat pressure trend as may occur during dipyridamole induced vasodilatation. A new concept of non-invasive blood pressure measurement by heart sound pattern analysis is described. The known diagnostic criterion of the 'accentuated' second heart sound of a hypertensive patient is here converted into a computer-aided pattern-recognition process for the second heart sound, applicable over the entire range of blood pressure. The method is in principle suited for automatically repeated blood pressure measurements, but further development is still needed for conversion into a widely practicable procedure. Integrating first heart sound [1], second heart sound amplitude and first-second heart sound time delay [3], a cutaneous operator-independent force sensor describes in real time systolic elastance, diastolic time, and systemic pressure trend, offering a new chance to monitor failing hearts.AbbreviationsA2: aortic component of the second heart sound; BSA: body surface area; C: systemic arterial compliance; DBP: diastolic blood pressure; CAD: coronary artery disease; CO: cardiac output; DCM: idiopathic dilated cardiomyopathy; EaI: effective arterial elastance index; EDV: end-diastolic volume; EF: ejection fraction; ESV: end-systolic volume; FFR: force-frequency relation; g: acceleration unit (9.8 m/sec2); HR: heart rate; LV: left ventricle/ventricular; LVMI: left ventricular mass index; M1: mitral component of the first heart sound; P2: pulmonary component of the second heart sound; S1: first heart sound; S2: second heart sound; SBP: systolic blood pressure; SVR: systemic vascular resistance; WMSI: wall motion score index.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsTB conceived this study, performed the data analysis, and drafted the manuscript; LV, CP, EPa, LP and MP were responsible for data collection and revised the manuscript; VG, EB, FF and MG were responsible for technology development and digital signal processing; GA gave a contribution to data discussion; EPi gave a contribution to preparation of study design, data discussion, and critical revision of the manuscript.Supplementary MaterialAdditional file 1Appendix. Sound – Heart sounds – Accelerometer to measure peak heart sounds vibration amplitude – Wireless – Wireless telemedicine – Telemedicine is healthcare's new frontier.Click here for file\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2532689",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2532689\nAUTHORS: Hua Yang, Jie Cao, Lian-Qing Li, Xia Zhou, Qiu-Li Chen, Wen-Ting Liao, Zong-Mei Wen, Shao-Hua Jiang, Rong Xu, Jian-An Jia, Xin Pan, Zhong-Tian Qi, Wei Pan\n\nABSTRACT:\nBackgroundProtein A, protein G and protein L are three well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting.ResultsWe used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D) and PA(A) respectively, B2 domain of protein G (PG) and B3 domain of protein L (PL) for affinity selection with human IgG (hIgG), human IgM (hIgM), human IgA (hIgA) and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A)-PG and PA(A)-PL were obtained in hIgG (hIgG1-Fc) and hIgM (hIgA) post-selection populations respectively. In addition, the linking peptides among all PA(A)-PG and PA(A)-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A)-PG and PA(A)-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively.ConclusionIn this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A)-PG and PA(A)-PL, were obtained, and demonstrate the novel Ig binding properties.\n\nBODY:\nBackgroundBacterial immunoglobulin (Ig)-binding proteins (IBPs) are cell-anchored which can bind to specific sites on Ig of the host and mediate pathogenicity in host [1]. Protein A of Staphylococcus aureus (SpA), protein G of group C and G streptococci (SpG), and protein L of Finegoldia magna formerly Peptostreptococcus magnus are three well-defined IBPs. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. SpA comprises 524 amino acid residues with a molecular weight of 57 KD. The extracellular part of SpA contains a tandem repeat of five highly homologous IgG-binding domains designated (from the N terminus) E, D, A, B and C, each of which contains about 58 amino acid residues. The overall structures of these domains are all up-down three α-helixes and all five domains of SpA show Ig-binding abilities [2-4]. SpG (about 63 KD) is composed of 594 amino acid residues, containing 3 highly homologous Ig-binding domains identified as B1, B2 and B3 [5]. Each domain of SpG consists of two pairs of antiparallel β-sheets connected by a single α-helix [6,7]. Protein L is a 95 KD protein and contains 719 amino acid residues, with the Ig-binding activity located in five homologous repeats, Bl-B5, each comprising 72–76 amino acid residues. The fold of the Ig-binding domains of protein L is similar to that of the domains of SpG [8,9].Both SpA and SpG show high affinity for the interface between the second constant region of heavy chain (CH2) and CH3 domains (CH2γ–CH3γ) of IgG crystallizable fragment (Fc) [10]. In addition, SpA can bind to fragment of antigen binding (Fab) of a subset of Igs with variable region of heavy chains belonging to human VHIII family, so SpA is capable of binding to other types of Ig molecules besides IgG [11,12]. In contrast to SpA, SpG binds to Fab with the first constant region of Ig γ chain (CH1γ), so SpG can bind only to IgG [13]. Protein L has been shown to bind to κ light chains of Ig, so it can bind to all types of Ig molecules that contain a kappa light chain [14,15].Some hybrid IBPs, like protein AG, Protein LG and Protein LA had been constructed [16-18], and were shown to sustain Ig-binding property of parental proteins and broaden Ig-binding spectra. Protein AG was shown to possess higher affinity for IgG than SpA or SpG. In addition to the enhanced affinity for IgG and IgG-Fc, protein LA showed enhanced affinity for Ig Fab or majority of human single chain variable antibody fragment (scFv) carrying the κ light-chain variable domain or expressing the VHIII determinant. It suggested complementary effect of Ig-binding in these hybrid IBPs.The single domains of SpA, SpG and protein L were all demonstrated to have function to bind to Ig [17,19-21]. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding remain interesting. In this study, we used the single Ig-binding domains of SpA, SpG and protein L as basic functional units to construct a combinatorial phage library displaying randomly-rearranged molecules joined by various random linking peptides, and conducted evolutional selection in vitro with four kinds of Ig molecules as bait. Two kinds of novel combinations of Ig-binding domains, PA(A)-PG and PA(A)-PL, were obtained, and might represent improved Ig-binding properties.ResultsDistribution of various fragment sizes displayed by phage library and post-selection populationsTo evaluate the Ig affinity selection efficacy, some markers including phage library binding capacity, output/input ratio of phages, distribution of various fragment size etc. were measured. The library binding capacity and output/input ratio did not correspond well with the affinity selection (data not shown). We found the distribution of fragment sizes changed remarkably during the selection (Fig. 1). As figure 2 shows, the proportion of phage clones displaying two domains and three domains in original library was 22%, then increased dramatically and reached 80%–100% after four rounds of selection with hIgG and recombinant hIgG1-Fc (Fig. 2A, 2B). In hIgM and hIgA selection, the proportion of phage clones displaying two domains and three domains also increased remarkably from 22% to 98% and 22% to 96% respectively after three or four rounds of selection (Fig. 2C, 2D). These results corresponded well with our previous experiment that also indicated that along with the rounds of selection, the proportion of phage clones displaying large fragments increased [22], and it might represent effective selection.Figure 1Detection of inserted fragments of phage clones in each round of hIgG selection library by PCR. PCR products were analyzed by electrophoresis in 1.2% agarose gel and detected by staining with ethidium bromide. No. 1 to 22: randomly picked phage clones; M: DL2000 Marker; C: positive control (pCANTAB5S vector); B: negative control (blank culture medium); I, II, III, IV: the first to the fourth round of selection respectively.Figure 2Proportion of phage clones with different sizes of inserted fragments in 22 phage clones after each round of selection with four Ig molecules respectively (A-D). : phage clones with no inserted fragment; : phage clones displaying one domain of combinatorial Ig-binding molecules; : phage clones displaying two domains of combinatorial Ig-binding molecules; : phage clones displaying three domains of combinatorial Ig-binding molecules.Analyses of inserted fragments of the post-selection populationsIn the fourth post-selection population selected with hIgG or hIgG1-Fc, twenty phage clones were randomly chosen for sequencing analysis. It was very interesting that the twenty sequenced phage clones from hIgG and hIgG1-Fc selection populations displayed the same combinations, all containing PA(A)-PG with different linking peptides (Table 1). Interesting results were also found about the distribution of random linking peptides. The different linking peptides showed divergent distribution in hIgG and hIgG1-Fc fourth post-selection populations. Of six different linking models, only PA(A)-PG (the second column in Table 2) existed in both hIgG and hIgG1-Fc post-selection populations, the other five PA(A)-PG combinations (from the third to seventh columns in Table 2) with different linking peptides existed in hIgG or hIgG1-Fc post-selection population.Table 1Sequence analyses of inserted fragments on phage clones in the original library and the third or fourth post-selection libraries with four Ig moleculesPhage LibrariesComposition of single domains of inserted fragmentThe original phage library (5*)PG-PL9N; PA(A)-PG-PL6N; PG3NR-PGR-PA(D)6N; PL3N-PA(D); PG6N-PA(A)3N-PLRThe 4th round of selection with hIgG (10)PA(A)-PG9N(5**);PA(A)-PG(2); PA(A)-PG3N(2); PA(A)6N-PG3NThe 4th round of selection with hIgG1-Fc (10)PA(A)-PG(7); PA(A)6N-PG(2); PA(A)3N-PG-PA(A)The third round of selection with hIgM (11)PA(A)-PL-PL9N(4); PA(A)6N-PL9N(2); PA(A)-PL3N(3); PA(A)9N-PL3N; PA(A)9N-PLThe 4th round of selection with hIgA (10)PA(A)-PL-PL9N(5); PA(A)6N-PL9N(2); PA(A)-PL3N(2); PA(A)9N-PL3N*: Number of sequenced phage clones; **: number of phage clones with the same inserted fragment; PA(A) and PA(D): A and D domains of protein A; PG: B2 domain of protein G; PL: B3 domain of protein L; 3N, 6N and 9N: the sequence of random linking peptides composed of three, six and nine nucleotides; R: reverse sequence of original sequence.Table 2Sequences of random linking peptides in PA(A)-PG structure and their distribution in hIgG and hIgG1-Fc selected librariesCombinatorial form of single domainsPA(A)-PGPA(A)-PG9NPA(A)-PG3NPA(A)6N-PG3NPA(A)6N-PGPA(A)3N-PG-PA(A)Nucleotide sequence of random linking peptide-AGC TTA CACCACACC TCG ACCCAC TCACCAAmino acid sequence of random linking peptide-S L HHT S TH SPNumber of clones Selected with hIgG (10*)252100Number of clones Selected with hIgG1-Fc (10)700021*: Number of sequenced phage clones; Underlined and italic parts represent the nucleotide and amino acid sequences of linking peptide followed the second domain. 3N, 6N and 9N: the sequence of random linking peptides composed of three, six and nine nucleotides. Single letter abbreviations of amino acids: H, His; L, Leu; P, Pro; S, Ser; T, Thr.Similar to the situation in hIgG or hIgG1-Fc selection, the displayed molecules in hIgA fourth post-selection population showed the same combinations with that in hIgM third post-selection population, all containing the PA(A)-PL (Table 1). In the twenty one randomly picked sequenced clones, twelve displayed PA(A)-PL and nine displayed PA(A)-PA(A)-PL (Table 1). In contrast with the results of hIgG and hIgG1-Fc selection populations, the sequences of linking peptides among PA(A)-PL structures tended to show convergent distribution. Almost all (4 of 5) combinations with different linking peptides existed in both hIgA and hIgM selection populations (from the second to fifth columns in Table 3), with an exception of PA(A)9N-PL (the sixth column in Table 3).Table 3Sequences of random linking peptides in PA(A)-PL structure and their distribution in hIgM and hIgA selected librariesCombinatorial form of single domainsPA(A)-PL-PL9NPA(A)6N-PL9NPA(A)-PL3NPA(A)9N-PL3NPA(A)9N-PLNucleotide sequence of random linking peptideTAC TGG TTGAAA CTA GCT AAC AACTTGGGT GAG ATG CACGAC TTT ATTAmino acid sequence of random linking peptideY W LK L A N NLG E M HD F INumber of clones selected with hIgM (11*)42311Number of clones selected with hIgA (10)52210*: Number of sequenced phage clones; Underlined and italic parts represent the nucleotide and amino acid sequences of linking peptide followed the second domain. 3N, 6N and 9N: the sequence of random linking peptides composed of three, six and nine nucleotides. Single letter abbreviations of amino acids: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; W, Trp; Y, Tyr.The Ig binding properties of the representative phagesEight representative positive phage clones were chosen and tested for the Ig binding properties. As figure 3 shows, number 1 to 5 of phage clones displaying PA(A)-PG as well as SpA-phage (clone 9) possessed strong binding activity with hIgG and hIgG1-Fc, but showed little binding to hIgM or hIgA, though this binding may theoretically exist through PA(A) domain interacting with VHIII region. It was very interesting that clone 4 and 5, which were from hIgG post-selection population, showed even stronger binding to hIgG than SpA-phage as well as clone 1, 2 and 3 which were found in hIgG1-Fc post-selection population (Fig. 3A). Clone 1, 2 and 3 showed even stronger binding to hIgG1-Fc than SpA-phage as well as clone 4 and 5 which were found in hIgG post-selection population (Fig. 3B). This binding priority suggested that the linking peptides could affect the Ig-binding properties. Compared with SpA-phage and 2L-phage (clone 10), clone 6, 7 and 8 displaying PA(A)-PL from hIgM or hIgA post-selection populations showed remarkable enhanced binding to hIgM or hIgA (Fig. 3C, 3D) and weak binding activity with hIgG and hIgG1-Fc (Fig. 3A, 3B). As SpA-phage, clone 1 to 5 phages showed some weak binding to hIgM or hIgA. 2L-phage bound to hIgG, hIgM or hIgA obviously, but showed no binding to hIgG1-Fc.Figure 3Detection of the binding activity of representative phage clones with four Ig molecules respectively by ELISA (A-D). Each of Ig molecules (labeled on top of each graph) was coated on ELISA plates using 0.1 M NaHCO3 (pH 9.6). The amplified representative phages (2 × 1011 TU) were added to each well and Ig-bound phages were detected with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. The displayed sequence of each representative phages was: 1, PA(A)3N-PG-PA(A); 2, PA(A)6N-PG; 3, PA(A)-PG; 4, PA(A)-PG9N; 5, PA(A)6N-PG3N; 6, PA(A)9N-PL3N; 7, PA(A)-PL-PL9N; 8, PA(A)9N-PL; 9, SpA-phage (positive control); 10, 2L-phage (positive control); 11, pCANTAB5S-phage (negative control). Comparable data were obtained in three independent experiments.The Ig binding properties of the novel combinatorial moleculesTo test the binding properties of selected combinatorial molecules, these molecules were expressed as fusion proteins with thioredoxin using expression vector pET32a(+) and performed Western Blot. All the PA-PG combinations as well as SpA and SpG showed strong binding to hIgG (Fig. 4A) and hIgG1-Fc (Fig. 4B), and weak binding to hIgM (Fig. 4C) and hIgA (Fig. 4D). Inconsistent with phage binding assays, the expressed PA-PG combinations did not show any binding advantage to hIgG (Fig. 4A) and hIgG1-Fc (Fig. 4B) compared with SpA and SpG. In contrast to PA-PG, the expressed PA-PL combinations showed much stronger binding to hIgM (Fig. 4C) and hIgA (Fig. 4D) than 4L and SpA respectively, which was consistent with phage binding assay.Figure 4Binding activities of seven fusion proteins of the novel combinatorial molecules, SpA, SpG and 4L with hIgG (A), hIgG1-Fc (B), hIgM (C) and hIgA (D) respectively by Western Blot. Seven fusion proteins, SpA, SpG and 4L (each of 5 μg) were separated by electrophoresis in SDS-PAGE and electrotransferred to nitrocellulose membrane respectively. The membrane was incubated with biotin labeled hIgG, hIgG1-Fc, hIgM and hIgA in 1:3 000 dilution respectively and detected with HRP-conjugated streptavidin, followed by developing with DAB. 1, fusion protein PA(A)3N-PG-PA(A); 2, fusion protein PA(A)6N-PG; 3, fusion protein PA(A)-PG; 4, fusion protein PA(A)-PG9N; 5, fusion protein PA(A)6N-PG3N; 6, fusion protein PA(A)9N-PL3N; 7, fusion protein PA(A)9N-PL; 8, fusion protein 4L; 9, SpA; 10, SpG.The PA(A)-PG or PA(A)-PL combinations showed binding advantagesTo test whether PA(A)-PG or PA(A)-PL combinations possess some binding advantages, we expressed the fusion proteins of PA(A)3N-PG-PA(A), PA(A)6N-PG from hIgG1-Fc post-selection population, PA(A)-PG9N, PA(A)6N-PG3N from hIgG post-selection population, and PA(A)9N-PL3N and PA(A)9N-PL from hIgM/hIgA post-selection population to perform competitive inhibition experiments. As figure 5 shows, all four PA(A)-PG combinations inhibited the binding of PA(A)-PG-phages to hIgG more efficiently than SpA and SpG alone (Fig. 5A, 5B, 5C, 5D). Furthermore, PA(A)-PG9N and PA(A)6N-PG3N from hIgG post-selection population showed more efficient inhibition than SpA, SpG and that both (Fig. 5C, 5D), while all four PA(A)-PG combinations inhibited the binding of SpA-phages to hIgG as efficiently as SpA, SpG and that both (Fig. 5E). Similar results were obtained for inhibition of PA(A)-PG-phages binding to hIgG1-Fc by competitive inhibition tests as above (data not shown). Consistent with phage binding tests (Fig. 3), the competitive inhibition experiments documented that the PA(A)-PG combinations possess some binding advantages to hIgG or hIgG1-Fc.Figure 5Competitive inhibition of PA(A)-PG-phages (A-D) and SpA-phage (E) binding to hIgG by PA(A)-PG combinations, PA(A)-PL combinations, SpA, SpG and both SpA plus SpG. 109TU of tested phages without and with each of 100 nM (black bars) or 25 nM (white bars) of inhibitor proteins were added into hIgG-coated wells respectively. Unbound phages were removed and 10 μl of exponentially growing E. coli TG1 was added into each well, incubated for 1 h at 37°C. The TG1 cells were harvested respectively and spread LB plates containing 100 μg/ml ampicillin, and bacterial colonies were counted after incubating at 37°C overnight. Inhibition rate was calculated: [1 - (mean of the bacterial colonies from tested wells with inhibitor proteins - mean of the bacterial colonies from blank control wells) divided by (mean of the bacterial colonies from tested wells without inhibitor proteins - mean of the bacterial colonies from blank control wells)] × 100%. No. 1 to 6: The expressed fusion proteins of PA(A)3N-PG-PA(A), PA(A)6N-PG, PA(A)-PG9N, PA(A)6N-PG3N, PA(A)9N-PL3N and PA(A)9N-PL were used as inhibitors respectively. No. 7 to 9: SpA, SpG and that both were used as inhibitors respectively.For PA(A)-PL-phages competitive inhibition experiments, expressed PA(A)-PL combinations inhibited the binding of PA(A)-PL-phages to hIgM much more efficiently than 4L, SpA alone and that both (Fig. 6A, 6B), while all PA(A)-PL combinations inhibited the binding of SpA-phage (Fig. 6C) or 2L-phage (Fig. 6D) to hIgM as efficiently as SpA, SpA and 4L or 4L, 4L and SpA respectively. Similar results were obtained for inhibition of PA(A)-PL-phages binding to hIgA by competitive inhibition tests as above (data not shown). Consistent with phage binding tests (Fig. 3), the competitive inhibition experiments showed the PA(A)-PL combinations possess obvious binding advantage to hIgM or hIgA.Figure 6Competitive inhibition of two representative PA(A)-PL-phages (A and B), SpA-phages (C) and 2L-phages (D) binding to hIgM molecules by PA(A)-PL combinations, PA(A)-PG combinations, SpA, 4L and both SpA plus 4L. 109TU of PA(A)-PL-phages, 1011TU of SpA-phages and 1010TU of 2L-phages without and with each of 100 nM (black bars) or 25 nM (white bars) of inhibitor proteins were added into hIgM-coated wells respectively. Unbound phages were removed and 10 μl exponentially growing E. coli TG1 was added into each well, incubated for 1 h at 37°C. The TG1 cells were harvested respectively and spread LB plates containing 100 μg/ml ampicillin, and bacterial colonies were counted after incubating at 37°C overnight. Inhibition rate was calculated: [1 - (mean of the bacterial colonies from tested wells with inhibitor proteins - mean of the bacterial colonies from blank control wells) divided by (mean of the bacterial colonies from tested wells without inhibitor proteins - mean of the bacterial colonies from blank control wells)] × 100%. No. 1 to 6: The expressed fusion proteins of PA(A)3N-PG-PA(A), PA(A)6N-PG, PA(A)-PG9N, PA(A)6N-PG3N, PA(A)9N-PL3N and PA(A)9N-PL were used as inhibitors respectively. No. 7 to 9: SpA, 4L and that both were used as inhibitors respectively.DiscussionCompared with SpA-phage displaying five domains of SpA, phages displaying PA(A)-PL which was contained in each sequenced clone as predominant combinations in hIgM and hIgA post-selection populations exhibited a remarkable enhanced binding affinity for hIgM and hIgA (Fig. 3). The prokaryotic expressed PA(A)-PL combinations also showed the same binding properties (Fig. 4C, 4D). Protein L binds primarily to κ light chains of I, III, IV subtypes of Igs [8], while SpA binds about 22% hIgA and 40% hIgM through interacting with VHIII region [11,23]. The coexistence of single domains of SpA and protein L could broaden the Ig-binding spectra, and achieve the binding advantage of PA(A)-PL for hIgM and hIgA. However, the loss of other possible combinations, like PL-PL, which should have same chance to be produced in original library, and may produce enhanced affinity for κ light chains due to avidity effect, suggested that PA(A)-PL should have additional binding advantage. Considering the binding properties of protein L and SpA and the structure of Ig-Fab, we speculated that the binding advantage of PA(A)-PL might be produced through double-site binding to VHIII and Vκ regions of Fab in hIgM and hIgA. In Fab fragment of hIgM and hIgA, the conformation of VH-VL is tightly fixed due to the interchain disulfide bond between VH and VL regions and non covalent interaction of VH-VL interface [24]. Moreover, the binding sites of protein L and SpA on Ig-Fab located on the opposite surface of the antigen binding cleft, and both interactions produce little steric hindrance to each other [15,25]. These characteristics are in favor of the double site binding of PA(A)-PL to VHIII and Vκ regions. This speculation was clearly supported by results of competitive inhibition experiments which showed that 4L, SpA alone or that both couldn't inhibit the binding of PA(A)-PL-phages to hIgM or hIgA as efficiently as PA(A)-PL combinations (Fig. 6A, 6B).It is predictable that PA(A)-PG combinations would be selected in hIgG and hIgG1-Fc post-selection populations. However, it was unexpected that PA(A)-PG was so predominant while PA(A/D)-PA (A/D) or PG-PG which had similar binding potential and same chance to be produced in original library was not selected (Table 2). This result suggests that PA(A)-PG combinations possess an advantage over other combinations in binding to Fc regions. It was supported by the phage binding assay which showed that the PA(A)-PG-phages selected by hIgG or hIgG1-Fc exhibited stronger binding to hIgG or hIgG1-Fc respectively than SpA-phages (Fig. 3A, 3B) and by the competitive inhibition test which showed that PA(A)-PG combinations inhibited the binding of PA(A)-PG-phages to hIgG or hIgG1-Fc more efficiently than SpA alone or SpG alone (Fig. 5A, 5B, 5C, 5D). The conformation of Fc was documented flexible, mobile and easy affected [26]. X-ray crystal structures studies for Fc and Fc-ligand complex indicated that the hinge proximal region of CH2 domain is disordered, suggesting internal mobility, generating a dynamic equilibrium between multiple conformers [27]. Interchange between heavy and light chain, binding to antigen and change of primary amino acid sequences of IgG (different IgG subtypes) would affect the Fc conformation [28-30]. Although SpA shares a lot of binding area in IgG-Fc with SpG, obvious difference between these two interactions was observed [31,32]. First, in SpG: Fc and SpA: Fc complex, the two helices in SpA domain are located mostly in CH2 side of Fc, the helix of SpG lies wedged in the CH2-CH3 cleft. Second, SpG interacts with Fc mainly through hydrogen bond, while SpA through hydrophobic interaction. Third, Fc has a set of unique amino acids for binding to SpG and SpA respectively [26]. So, although the binding sites of SpA and SpG overlap, their binding nature is different, and the structure of PA(A)-PG could produce the different binding avidity for a pair of Fc sites in one hIgG molecule from that produced by PA(A/D)-PA (A/D) or PG-PG, which was documented to possess some binding advantage, and therefore showed the selection advantage.In this work, the proportion of phage clones displaying two and three domains also increased remarkably along with the rounds of selection (Fig. 1, Fig. 2), and the linking peptides were significantly selected (Table 2, Table 3). These results might reflect the effectiveness of the selection and the significance of selected PA(A)-PG and PA(A)-PL. The conformation of binding sites for IBPs of hIgA and hIgM Fab were fixed and stable, as well as the linking peptide among all selected PA(A)-PL structures showed some convergent distribution. Different from the native hIgG, the Fab of hIgG1-Fc was substituted by TNF receptor. It could produce some conformation difference between hIgG1-Fc and native hIgG, and could be responsible for the divergent distribution of linking peptide in hIgG and hIgG1-Fc post-selection populations. The phage binding assay and competitive inhibition test also showed comparable binding advantage for the clones from selection hIgG population with hIgG, and for those from hIgG1-Fc post-selection population with hIgG1-Fc (Fig. 3, Fig. 5C, 5D). This result suggested that the combinations and special linkage of the different IBP domains could sensitively reflect the conformational change in the binding sites of Ig Fc.ConclusionIn this study, a combinatorial phage library displaying single domain randomly-rearranged molecules derived from natural bacterial IBPs was selected with hIgG, hIgM, hIgA and hIgG1-Fc. Two kinds of novel combinations of Ig-binding domains, PA(A)-PG and PA(A)-PL, which don't exist in natural bacterial Ig-binding molecules, were obtained, and showed the comparable binding advantages. It demonstrated the novel binding properties.MethodsSingle domain randomly-rearranged combinatorial phage displayed libraryThe construction of the phage library was described previously [33]. Briefly, gene fragments encoding A and D domains of SpA [PA(A) and PA(D)], B2 domain of SpG (PG) and B3 domain of protein L (PL) were individually generated by PCR amplification using the primers (Table 4) which introduced recognition site for Xba I in both ends of the fragments and nucleoside acid sequences in the 3'-end, which encoding random linking peptide consisted of 0, 1, 2 or 3 amino acids. Then the PCR products were digested with Xba I and ligated into the Xba I site of the phagemid pCANTAB5S to construct a phage displayed random combinatorial library. The library has size of 2 × 107 members, and titer of the phage library is calculated to 1.3 × 1011 transformation unit (TU)/ml. Host bacterial strain TG1 was from Stratagene Company, Cambridge, England. Primers located in the upward and downward of the cloning site of vector pCANTAB5S were used to amplify the inserted fragment of positive phages and to perform sequencing analysis of inserted fragment. Both of forward primer designated P1: 5'-CAA CGT GAA AAA ATT ATT ATT CGC-3' and reverse primer designated P2: 5'-GTA AAT GAA TTT TCT GTA TGA GG-3' was obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.Table 4Primers for amplification of DNA fragments encoding each Ig-binding domainsNameDescriptionSequence(5' → 3')PA(A)-UxkSense terminal primer of PA(A)C CTG GGT ACCTCT AGA* GCT GAC AAC AAC TTC AACPA(D)-UxlSense terminal primer of PA(D)TAT GGT ACC TCT AGA GCT GAC GCT CAG CAG AACPA(A/D)-DxkAntisense random primer of PA(A/D)ACT GGT ACC TCT AGA (0N, 3N, 6N, 9N)** TTT CGG AGC CTG AGA TTCPG-UxkSense terminal primer of PGGCG GGT ACC TCT AGA ACC TAC AAA CTG GTT ATCPG-DxkAntisense random primer of PGTCA GGT ACCTCT AGA (0N, 3N, 6N, 9N) TTC GGT AAC GGT GAA GGTPL-UxkSense terminal primer of PLGCG GGT ACCTCT AGA AAA GAA AAA ACC CCG GAAPL-DxkAntisense random primer of PLTGC GGT ACC TCT AGA (0N, 3N, 6N, 9N) ACC AGC GAA TTT GAT GTT CAG*: Italic and black parts represent Xba I recognition sites; **: Underlined parts represent the random linking sequence; 0N, 3N, 6N and 9N: the sequence of random linking peptides composed of non, three, six and nine nucleotides.Vectors and reagentsPhagemid vector pCANTAB5S and phage displaying E-D-A-B-C domains of SpA (SpA-phage) were constructed by our lab and has been described previously [34]. Briefly, phagemid pCANTAB5S was obtained by inserting the DNA fragment of Xba I-Stu I-Sal I-Kpn I-(Gly4Ser)3 into pCANTAB5L (Pharmacia Biotech, Uppsala, Sweden) between Sfi I and Not I cloning sites. The encoding sequence of SpA [35] was inserted into pCANTAB5S at Stu I site to construct SpA-phage. 2L-phage containing two domains (B3-B3) of protein L was obtained from a phage library displaying Ig-binding mono-domains of SpA and protein L by affinity selection with hIgG [22]. Human IgG (hIgG), human IgM (hIgM), human IgA (hIgA) and SpG were from Sigma, St. Louis, MO, USA. Prokaryotic expressed SpA (Genbank: P02976) and hIgG1-Fc molecule that is obtained through substituting Fab of human IgG1 with soluble receptor of human tumor necrosis factor (TNF) by gene engineering were kindly provided by Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co. Ltd, Shanghai, China. All antibodies were biotinylated using biotinyl-N-hydroxy-succinimide (Pierce, Rockford, IL, USA). Purified protein 4L containing four domains (B3-B3-B3-B3) of protein L was expressed by using prokaryotic expression vector pET32a(+) in E. coli BL21 following the protocol provided by Novagen Company (Germany) and purified by Ni-NTA column (Pharmacia Biotech) at our lab (data not shown). Helper phage M13K07 and horseradish peroxidase (HRP)-conjugated anti-M13 antibody were from Pharmacia Biotech, Uppsala, Sweden.Selections of the phage displayed library with four Ig moleculeshIgG, hIgM, hIgA and hIgG1-Fc molecules were diluted in coating buffer (0.1 M NaHCO3, pH 9.6) resulting in 10 μg/ml respectively and coated in sterile 96-well ELISA plates at 37°C for 3 h. After blocking the plates with blocking buffer (10% degreased milk powder, 0.1% Tween 20 and 0.2% mercurothiolate in 0.01M phosphate-buffered saline) for at least 1 h, phage displayed library (about 1010–11 TU) was added into each well and incubated for 3 h at 37°C. Unbound phages were removed by washing with washing buffer (0.25% Tris, 0.05% Tween 20 in ddH2O) 30 times with vigorous pipetting, and 100 μl E. coli TG1 at an optical density at 600 nm of about 0.5 were added into wells, incubated for 1 h at 37°C. The number of eluted phages was calculated by colony forming units (c.f.u.) on TG1 cells in LB plates containing 100 μg/ml ampicillin. E. coli TG1 cells harboring eluted phage were amplified for 1 h by shaking 250 rpm at 37°C in 8 ml LB medium. Then ampicillin (100 μg/ml) and helper phages M13K07 (about 3 × 1012 TU) were added and the E. coli TG1 cells were cultured as above. After 1 h, kanamycin (50 μg/ml) was added and the E. coli TG1 cells were grown continuously by shaking 180 rpm at 37°C overnight. Phages were harvested by centrifugation (10 min, 5000 × g) of the medium and filtration of supernatant through 0.22 μm filter membrane, then used for the subsequent round of selection with the same bait. Three or four rounds of selection were performed as above.Detection of distribution and size of inserted fragments in primary library and each round post-selection population by PCRTwenty two phage clones in primary library and each round post-selection population were picked randomly and cultured in 0.5 ml LB medium by shaking 250 rpm at 37°C for 5 h respectively. The culture medium was used as template to amplify inserted fragments in these phages by PCR. For DNA amplification, 1 μl of template was added to a 50 μl reaction mixture containing 5 μl of 10× reaction buffer (500 mM KCl, 100 mM Tris-HCl pH 9.0, 1% Triton X-100), 1 μmol of each primer (P1 and P2), 3 mmol Mg++, 100 μmol dNTP, 1 U Taq DNA polymerase (2 U/μl, Promega, Madison, WI, USA) and nuclease-free water. The reaction mixture was amplified on a thermocycler (Perkin Elmer Applied Biosystems, USA) for 30 cycles of 30 s at 94°C, 30 s at 50°C, and 45 s at 72°C followed by a 5 min extension at 72°C. PCR products were analyzed by electrophoresis in 1.2% agarose gel and detected by staining with ethidium bromide. pCANTAB5S phagemid and blank culture medium were used as template for positive and negative controls respectively.Sequence analysesFive to eleven positive phage clones identified by PCR were picked randomly from the primary phage library and the third or fourth post-selection populations. Inserted DNA fragment of positive phages were sequenced using the primers P1 and P2. Corresponding amino acid sequences were deduced from DNA sequences and a multiple sequence alignment was analyzed with the DNASTAR software package.Detection of representative positive phages binding to Ig molecules by ELISA10 μg/ml each of Ig molecules (hIgG, hIgM, hIgA or hIgG1-Fc) was coated on ELISA plate as described above. About 2 × 1011 TU amplified representative positive phages obtained from the each round of selection were added to each well and then incubated for 2 h at 37°C. The wells were washed with phosphate-buffered saline (PBS) containing 0.05%Tween 20 and the bound phages were detected with HRP-conjugated anti-M13 phage antibody. The development was performed by the addition of diaminobenzidine (DAB) (Sigma, St. Louis, MO, USA), and read at 490 nm in an ELISA Reader (Bio Rad). SpA-phages and 2L-phages were used as positive controls respectively. The pCANTAB5S-phage (obtained by infecting E. coli TG1 with blank phagemid pCANTAB5S) was used as a negative control.Expression of the novel combinatorial moleculesBacterial clones harboring positive phagemids displaying five PA(A)-PG combinations and two PA(A)-PL combinations were used as template respectively to amplify DNA fragments by PCR using forward primer (5SNco-u) and reverse primer (5SNoG-d). The forward primer 5SNco-u contained Nco I recognition site (Table 5). The synthetic primers were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. For DNA amplification, the composition in 50 μl reaction mixture was the same as above. The reaction mixture was amplified on a thermocycler (Perkin Elmer Applied Biosystems, USA) for 35 cycles of 30 s at 94°C, 30 s at 60°C, and 45 s at 72°C followed by a 5 min extension at 72°C. The amplified DNA fragment containing a BamH I cloning site of the phagemid at 3' terminal of displayed sequence was digested with Nco I and BamH I and inserted into the Nco I-BamH I site of prokaryotic expression vector pET32a(+) (Qiagen, Valencia, CA). The recombinant plasmid was identified and sequenced by forward sequencing primer (B-S-U) and reverse sequencing primer (S-H-D) (Table 5). Competent E. coli BL21(DE3) cells were transformed using above positive recombinant plasmid mediated by CaCl2 and spread on LB plates (containing 100 μg/ml of Amp and 15 μg/ml of Kana), and cultured at 37°C overnight. Transformed positive BL21(DE3) colony was picked up and cultured in 500 ml LB medium by shaking 250 rpm at 37°C with ampicillin (100 μg/ml). The Log-phase bacteria were induced expression by adding 500 μl of 1 M isopropyl-beta-D-thiogalactopyranoside (IPTG) in the medium and continuously cultured for 3 h. The pellet cells were collected after centrifugation at 6 000 rpm for 10 min at 4°C and washed by PBS (pH 7.2). Then the cells were resolved by using 8 M urea (pH 8.0) and Ni-NTA column (Amersham Pharmacia Biotech) was used to purify the expression proteins. The purified proteins were dialyzed thoroughly against PBS (pH 7.0). The concentrations of the proteins were detected by routine Bradford assay.Table 5Primers for amplifying exogenous DNA sequences of selected representative phagesNameDescriptionSequence (5' → 3')5SNco-uForward amplifying primerTATCCATGG*CTGCGGCCCAGCCGGCCTCT5SNoG-dReverse amplifying primerCCTGCGGCCGCAACTGCCGCCGCCB-S-UForward sequencing primeGGA TCC GAG CTC AGG CCT GTC GAC GGT ACC GTTS-H-DReverse sequencing primerGAG CTC AAG CTT ACC AGA TCC ACC ACC GCC GGT ACC*: Underlined part represents Nco I recognition site.Competitive inhibition testIn order to avoid experimental results being interfered due to binding of SpA-phage and other phages displaying more than two IBP domains to conjugated secondary antibodies, the competitive inhibition tests were established in this study by using E. coli TG1 infection as a substitute for HRP-conjugated anti-M13 phage antibody detection. Briefly, 1 μg each of Ig molecules (hIgG, hIgG1-Fc, hIgA or hIgM) were coated on sterile 96-well microtitration plates by using 0.1 M NaHCO3 (pH 9.6) at 37°C for 3 h. After blocking the plates with blocking buffer (10% degreased milk powder, 0.1% Tween 20 and 0.2% mercurothiolate in 0.01 M phosphate-buffered saline) for at least 1 h, the tested phages without and with 100 nM or 25 nM of inhibitor proteins were added into hIgG-coated or hIgG1-Fc-coated wells respectively, and incubated at 37°C for 1 h. Unbound phages were removed by washing with washing buffer (0.25% Tris, 0.05% Tween 20 in ddH2O) 10 times with vigorous pipetting after incubating for 3 h at 37°C, and 10 μl E. coli TG1 at an optical density at 600 nm of about 0.2 was added into each well, incubated for 1 h at 37°C. The infected TG1 cells of each well were harvested respectively and spread LB plates containing 100 μg/ml ampicillin, and bacterial colonies were counted after incubating at 37°C overnight. Three parallel wells of each test were detected, and the mean of bacterial colonies from each test wells was used to calculate the inhibition rate. Inhibition rate wascalculated: [1 - (mean of the bacterial colonies from tested wells with inhibitor proteins - mean of the bacterial colonies from blank control wells) divided by (mean of the bacterial colonies from tested wells without inhibitor proteins - mean of the bacterial colonies from blank control wells)] × 100%.Western BlotEach of 5 μg of the tested combinatorial IBP molecules, SpA, SpG and 4L were separated by electrophoresis in sodium dodecyl sulphate-polyacrylamid gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose membrane (Millipore, Pharmacia) respectively. The membrane was blocked with blocking buffer (10% degreased milk powder, 0.1% Tween 20 and 0.2% mercurothiolate in 0.01 M PBS) at 4°C over night. After washing with PBS containing 0.05% Tween 20, the membrane was incubated with labeled hIgG (1 mg/ml, 1:3 000) at 37°C for 2 h. The membrane was washed with PBS containing 0.05% Tween 20 for 6 times and detected with HRP-conjugated streptavidin, followed by developing with diaminobenzidine (DAB). The interactions of the tested proteins with hIgG1-Fc, hIgM or hIgA were parallel detected by Western Blot respectively as above.Authors' contributionsHY, JC and L–QL carried out the selections of the phage displayed library with four Ig molecules and drafted the manuscript. XZ, Q–LC and Z–MW performed the detection of distribution and size of inserted fragments in primary and each round post-selection population by PCR, and competitive inhibition test. HY and W–TL carried out expression of the fusion proteins and Western blot experiments. S–HJ, RX, J–AJ and XP performed the ELISA of selected positive phages and the sequence analyses. WP and Z–TQ conceived, designed and coordinated the original project. WP and JC wrote and revised the manuscript. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2532749",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2532749\nAUTHORS: Philippe A. Parone, Sandrine Da Cruz, Daniel Tondera, Yves Mattenberger, Dominic I. James, Pierre Maechler, François Barja, Jean-Claude Martinou\n\nABSTRACT:\nMitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.\n\nBODY:\nIntroductionMitochondria form a highly dynamic tubular network in eukaryotic cells. The organisation, shape and size of these organelles is regulated by movements along the cytoskeleton but also by frequent fission and fusion events [1], [2]. Evolutionary conserved cellular components that regulate mitochondrial fission and fusion have been identified in yeast, fly and mammals [3]. Mitochondrial fission relies on a large dynamin related GTPase called Drp1 (Dnm1p in yeast). Drp1 is located mostly in the cytosol of mammalian cells and a pool of the protein translocates to the mitochondrial tubules where it assembles, through its interaction with hFis1 [4], [5], into foci at future fission sites [6], [7]. Inhibition of Drp1 function using either expression of DrpK38A, a dominant negative mutant defective in GTP binding, or RNA interference, leads to the formation of a highly fused and tubular mitochondrial network, thus implicating Drp1 in mitochondrial fission [6], [8]. Mitochondrial fusion in mammalian cells depends on a distinct set of evolutionary conserved components, namely the dynamin-related GTPases Mfn1,2 and OPA1 (for reviews see [3]).Mitochondrial dynamics is clearly important in cellular homeostasis. Mutations in the OPA1 or MFN2 genes respectively cause the most commonly inherited optic and peripheral neuropathies (autosomal dominant optic atrophy and Charcot-Marie-Tooth disease; [9], [10]). Studies on cultured mammalian cells have shown that formation of a reticular mitochondrial network is important for proper mitochondrial calcium buffering and for propagating intra-mitochondrial Ca2+ waves [11], [12]. Mitochondrial fusion is required for the maintenance of mitochondrial DNA (mtDNA; [13]) and inhibiting this process has been shown to reduce the activity of the electron transfer chain (ETC; [14]) and to reduce mitochondrial metabolism [15]. The role of mitochondrial fission, on the other hand, is less clear. It has been proposed to be required for apoptosis [16], [17], although this proposal has recently been challenged [18]–[20].In this study, we set out to determine the role of mitochondrial fission in mitochondrial and cellular homeostasis. Here, we show that preventing mitochondrial fission by down-regulating expression of Drp1 leads to mitochondrial dysfunction, an increase in cellular reactive oxygen species (ROS) and a loss of mtDNA which correlates with a depletion of cellular ATP, inhibition of cell proliferation and autophagy.ResultsDepletion of Drp1 in HeLa cells leads to mitochondrial dysfunctionIn order to investigate the role of mitochondrial fission in mitochondrial and cellular homeostasis, RNA interference was used to down-regulate expression of Drp1. To this end, a small hairpin RNA (shRNA) targeting the Drp1 transcript was synthesised in vivo by means of the shRNA expression vector pRETRO-SUPER (D1; [21]). As a control, a similar construct expressing a shRNA targeting the luciferase transcript was used (Ctrl). As shown in Fig. S1A, protein levels of Drp1 were strongly reduced at 96 h after transfection of HeLa cells with the D1 construct. At the same time point, analysis of mitochondrial morphology by immunofluorescence using an anti-TOM20 antibody, revealed highly fused and interconnected mitochondria (Fig. S1B), confirming that Drp1 is required for mitochondrial fission [7].To assess whether mitochondrial fission is required for the maintenance of mitochondrial homeostasis, mitochondrial functional parameters were measured in Drp1-depleted cells using flow cytometry. Mitochondrial inner membrane potential (ΔΨm) is a critical aspect of mitochondrial homeostasis. We therefore determined if ΔΨm was affected in Drp1-depleted cells by quantifying fluorescence of the cationic dye JC-1 by flow cytometry.JC-1 indicates mitochondrial polarization by shifting its fluorescence from green (FL1; ∼525 nm) to red (FL2; ∼590 nm) in a potential-sensitive manner due to concentration-dependent formation of red fluorescent J-aggregates. As shown in Fig. 1A, ΔΨm (expressed as the ratio of FL2/FL1 in order to account for variations in mitochondrial volume) is significantly lower in Drp1-depleted cells, (58,8%±SEM 5.2, compared to Ctrl cells). The same results were obtained when a different potentiometric dye, namely TMRE, was used to determine ΔΨm (data not shown). We next investigated if the production of ROS was altered upon inhibition of mitochondrial fission. The levels of ROS were measured by flow cytometry in Drp1-depleted cells using carboxy-H2DCFDA which upon exposure to oxidative species is oxidized to the green fluorescent probe carboxy-DCF. As shown in Fig. 1B, Drp1-depleted cells loaded with carboxy-H2DCFDA emitted significantly more in the green (FL1) spectra (160.5%±SEM 19.8, compared to Ctrl cells). The ΔΨm and ROS levels were similarly decreased and elevated respectively when Drp1 was depleted in HeLa cells using an shRNA, D2, that targets a different region of the Drp1 transcript (Fig. S1A, B and Fig. 1A, B).10.1371/journal.pone.0003257.g001Figure 1Depleting HeLa cells of Drp1 leads to mitochondrial dysfuntion.A. HeLa cells were transiently transfected with the Ctrl, D1 or D2 constructs, selected with puromycin for 24 h, collected 96 h after transfection and stained with JC1 for flow cytometric analysis. The results are expressed as a percentage of the ratio between red and green emissions (FL2/FL1) in Ctrl cells and represent the mean+SEM from 7 independent experiments (***: P<0.0005). B. HeLa cells treated as in A. were stained with DCFDA for flow cytometric analysis. The results are expressed as a percentage of the DCFDA green fluorescence (FL1) in Ctrl cells and represent the mean+SEM from 7 independent experiments (*: P<0.05). C. HeLa cells were infected with Ctrl, D1 or D2 retroviruses, selected with puromycin for 24 h, and collected 144 h after the infection. Oxygen consumption was expressed as a percentage of the O2 consumption in Ctrl cells. The results represent the mean+SEM from 4 independent experiments (*: P<0.05). D. Oxygen consumption was assessed in mitochondria isolated from HeLa infected with the Ctrl, D1 or D2 retroviruses. The percentage uncoupling was determined by dividing the amount of oxygen consumption in state IV (in the presence of Succinate only) by that in state III respiration (in the presence of Succinate and ADP). The results represent the mean+SEM from 3 independent experiments (**: P<0.005). E. DNP-derivatized protein lysates of mitochondria isolated from Hela cells treated as in A were separated by polyacrylamide gel electrophoresis followed by Western blotting analysis using the indicated antibodies.Together these results suggest that the function of mitochondria in Drp1 depleted cells may be impaired and that oxidative damage in these organelles may be increased. We therefore examined the principal activity of the organelle, namely mitochondrial respiration, in D1 and D2 transfected cells using standard Clark electrode oxymetry. As shown in Fig. 1C, oxygen consumption in growth media (at pH 7.5) was significantly decreased in intact live Drp1-depleted cells (74.7%±SEM 6.1 and 72.7%±SEM 4.9 for D1 and D2 respectively compared to Ctrl cells). Furthermore, when respiration was assessed in isolated organelles, mitochondria from Drp1-depleted cells not only consumed less oxygen in the presence of succinate and ADP (state III; data not shown) but were also markedly uncoupled, as judged by the increased ratio of State IV (succinate without ADP) to State III (Fig. 1D). These results suggest that depleting cells of Drp1 causes mitochondrial dysfunction. To determine if this correlates with mitochondrial oxidative damage, we determined the level of protein carbonyls in mitochondria isolated from D1 and Ctrl cells through a reaction with 2,4-dinitrophenylhydrazine (DNPH). As shown in Fig. 1E, the level of protein oxidation in D1 mitochondria was 37% higher compared to Ctrl.Preventing mitochondrial fission in HeLa cells leads to a decrease in cellular ATP content, inhibition of cell proliferation and autophagyMitochondria are a major source of ATP in all cell types including HeLa cells, as inhibiting mitochondrial ATP production in these cells (using the ATP synthase inhibitor oligomycin) leads to a 50% decrease in total cellular ATP levels (Fig. 2A). Since mitochondrial respiration is significantly impaired in Drp1-depleted cells, we examined the levels of ATP in HeLa cells transfected with Drp1 RNAi. As shown in Fig. 2B, in D1 and D2 cells, ATP levels were reduced by approximately half (44.4%±SEM 7.7 and 53.7%±SEM 2.0 for D1 and D2 respectively) compared to Ctrl cells. Therefore, the mitochondrial dysfunction observed in cells depleted of Drp1 leads to a significant drop in total cellular ATP levels. Importantly, we have previously reported that depleting HeLa cells of Drp1 using the D1 or D2 constructs does not induce cell death (at the time points tested) when assessed by flow cytometric analysis of Annexin V and propidium iodide stained cells [18]. Therefore, since the drop in total cellular ATP at 96 hrs after transfection of HeLa cells with the D1 or D2 constructs was compatible with cell survival, we determined if it affected cell propagation. We investigated the rate of proliferation of Drp1-depleted cells by quantifying the uptake of the thymidine analog bromodeoxyuridine (BrdU), which is incorporated into newly synthesized DNA strands of actively cycling cells. As shown in Fig. 2C, the number of cells that incorporated BrdU was significantly decreased in D1 and D2 transfected cells compared to Ctrl (35.0%±SEM 7.0 for D1, 55.4%±SEM 1.0 for D2 and 83.4%±SEM 2.4 for Ctrl).10.1371/journal.pone.0003257.g002Figure 2Inhibiting mitochondrial fission in HeLa cells leads to drop in ATP levels and a proliferative arrest.A. HeLa cells were treated with the F1F0 ATP synthase inhibitor oligomycin (10 µM) and total cellular ATP levels were determined after 2 h of treatment. ATP levels after oligomycin treatment are expressed as the percentage of the ATP level in untreated HeLa cells. Results are calculated from 3 independent experiments+SEM (***: P<0.0005). B. HeLa cells were transiently transfected with the Ctrl, D1 or D2 constructs, selected with puromycin for 24 h, collected 96 h to measure total cellular ATP content. The quantity of ATP in D1 and D2 cells was expressed as a percentage of the ATP in Ctrl cells. The results represent the mean+SEM from 3 independent experiments (*: P<0.05). C. 96 h after transfection, Hela cells treated as in B. were grown in the presence of 10 µM BrdU for a further 18 h, fixed and stained with a BrdU antibody. The percentage of cells positively stained with BrdU was quantified from 3 independent experiments+SEM (***: P<0.0005).Autophagy is typically activated by fasting and nutrient deprivation [22]. Since preventing mitochondrial fission in HeLa cells led to depletion of ATP, we assessed if this was accompanied by an autophagic response in Drp1-depleted cells. Microtubule-associated protein light chain 3 (LC3) is a widely used marker to monitor autophagy. Upon the induction of autophagy LC3 relocalises to the newly formed autophagosomes, changing from a diffuse to a punctate pattern as observed by immunostaining, and is modified to a more rapidly migrating form that can be observed on SDS-PAGE [23]. As shown in Fig. 3A, the immunostaining pattern of D1 transfected cells with an anti-LC3 antibody was distinctively punctate compared to the diffuse staining pattern observed in Ctrl cells (45.2%±SEM 5.1 and 3.8%±SEM 1.7 of the D1 and Ctrl cells respectively had a punctate LC3 immunostaining pattern, Fig. 3B). Similar results were obtained in D2 cells (Fig. 3B). Furthermore, by immunoblotting the protein levels of the more rapidly migrating form of LC3 (induced upon upregulation of autophagy) were significantly increased in total lysates of Drp1-depleted cells compared to Ctrl cells (Fig. 3C). These results show that inhibition of mitochondrial fission in HeLa cells induces autophagy. Of note, from the evidence presented in Fig. 3A very few of the LC3 positive vesicles colocalise with the mitochondrial marker cytochrome c (arrowheads on merged Fig. 3A) and LC3 positive punctae colocalise only with small round mitochondria.10.1371/journal.pone.0003257.g003Figure 3Inhibiting mitochondrial fission in HeLa cells triggers autophagy.A. HeLa cells were transiently transfected with the Ctrl, D1 or D2 constructs, selected with puromycin for 24 h, fixed 96 h post-transfection and co-stained with antibodies against LC3 and cytochrome c. The scale bar corresponds to 15 µm. B. The number of cells in A. with a punctate LC3 pattern was quantified and the results represent the mean+SEM from 3 independent experiments (***: P<0.0005). C. Cells treated as in A. were collected 96 h after transfection for Western blotting analysis using the indicated antibodies.Preventing mitochondrial fission in HeLa cells leads to loss of mitochondrial DNAIt is now accepted that excessive mitochondrial fission, induced by a loss of fusion protein such as mitofusins or Fzo, leads to a loss of mtDNA in yeast and in mammalian cells [24], [25]. Therefore we set out to determine if inducing excessive fusion of the mitochondrial network by depleting HeLa cells of Drp1 led to alterations in the levels of mtDNA. Mitochondrial DNA nucleoids were stained with an anti-DNA antibody in D1 and Ctrl cells. As shown in Fig. 4A the DNA-specific antibodies labeled punctate structures that have been previously reported to correspond to mtDNA nucleoids [26]. These punctae are distributed throughout the entire mitochondrial network in Ctrl cells, as shown by co-staining with the outer-mitchondrial membrane marker TOM20. However, in Drp1-depleted cells most of the long tubular mitochondria were devoid of DNA punctae and several intensely stained mtDNA nucleoids were often clustered in discreet regions of the mitochondrial tubule or in large vesicular mitochondria close to the nucleus (see arrowheads in Fig. 4A). This later observation was confirmed when the average fluorescent intensity of individual mtDNA nucleoids was quantified and found to be 1.5 fold higher in D1 cells (Fig. 4B). These data show that in Drp1-depleted cells extensive portions of the mitochondrial network are devoid of mtDNA, or contain quantities of mtDNA that cannot be detected by immunostaining, and that the remaining mtDNA nucleoids often cluster together and contain more copies of the mtDNA molecules. In order to determine quantitatively whether there was a change in the total levels of mtDNA in Drp1-depleted cells, we used quantitative PCR amplification of the 12S ribosomal RNA small subunit mitochondrial gene as previously reported [26]. As shown in Fig. 4C, the levels of 12S rRNA gene were significantly lower in D1 cells (50.6%±SEM 9.1) compared to Ctrl cells. Depleting Drp1 from HeLa cells using the D2 shRNA also led to a similar decrease in mtDNA (by 51.9%±SEM 8.8). Altogether, these results suggest that inhibiting mitochondrial fission in HeLa cells leads to a loss of mtDNA. In Fig. 1 we provide evidence that, in HeLa cells depleted of Drp1, ROS levels and mitochondrial protein oxidation are significantly increased. In order to determine if oxidative damage is a cause of mtDNA loss in D1 cells we tested whether expression of a mitochondrially targeted form of catalase (mCAT) could reverse mtDNA depletion in D1 cells. Previous studies have shown that mitochondria isolated from mCAT transgenic animals produce less H2O2 and have lower levels of mtDNA oxidative damage [27]. As shown in Fig. 4D, the levels of 12S rRNA gene in HeLa cells cotransfected with D1 and mCAT were not significantly different from those in Ctrl cells. Altogether, these results suggest that inhibiting mitochondrial fission in HeLa cells leads to a loss of mtDNA and that this depletion can be prevented by the expression of an H2O2 scavenger at the mitochondria.10.1371/journal.pone.0003257.g004Figure 4Inhibiting mitochondrial fission in HeLa cells leads to a loss of mtDNA.A. HeLa cells were transiently transfected with the Ctrl, D1 or D2 constructs, selected with puromycin for 24 h, fixed 96 h post-transfection and stained with antibodies against DNA and TOM20. The scale bar corresponds to 15 µm. B. The integrated intensity of individual mtDNA nucleoids of the cells in A. was quantified from maximal projection images from three independent experiments and the results represent the intensity of 1824 nucleoids from 10 Ctrl cells and 584 nucleoids from 9 D1 cells. C. Cells treated as in A. were collected 96 h after transfection and DNA was extracted for quantitative PCR amplification of the 12S ribosomal RNA small subunit mitochondrial gene. The results are expressed as a percentage of the mtDNA in Ctrl cells and represent the mean+SEM from 3 independent experiments (*: P<0.05). D. HeLa cells transfected with the indicated constructs were treated as in A. and processed as in C. The results are expressed as a percentage of the mtDNA in Ctrl cells and represent the mean+SEM from two experiments (*: P<0.05, ns: not significant).DiscussionIn this study we report that depleting cells of Drp1 leads to mitochondrial dysfunction, an increase in cellular ROS levels and loss of mtDNA which is accompanied by a drop in cellular ATP levels, a proliferative arrest and autophagy.Recent studies have reported that mammalian cells depleted of Drp1 produced less ATP and consumed less oxygen [20], [28], thus supporting some of our conclusions. In addition we find that there is a significant increase in ROS levels, that mitochondrial respiration is markedly uncoupled and that mtDNA is lost when mitochondrial fission is inhibited by depletion of Drp1. This raises the question as to what is the primary cause of these mitochondrial alterations and in light of the results presented here we propose that Drp1 dependent fission protects the mitochondria from excessive damage. It is widely accepted that the vast majority of cellular ROS can be traced back to the mitochondria, therefore making this organelle a primary target for oxidative damage which has been shown to result in mitochondrial dysfunction and depletion of mtDNA [29]–[33]. Interestingly, it has been reported that inducing a localized increase in ROS levels along mitochondrial tubules results in a drop in ΔΨm in the targeted zone which is followed by the fragmentation of this portion of the mitochondrial network [34], [35]. In this study we show that preventing mitochondrial fragmentation leads to mitochondrial oxidative damage and mtDNA loss and that expressing a H2O2 scavenger at the mitochondria in D1 cells prevents the loss of mtDNA. Therefore, altogether these results suggest that mitochondrial fission is required to isolate (through fragmentation) damaged regions of the mitochondrial tubule. This would prevent accumulation of damage in the mitochondrial network and preserve the function, as well as the genome, of the organelle.Alternatively, the primary cause of the mitochondrial alterations observed in Drp1 deficient cells could be the loss of mtDNA, which would lead to loss of respiratory capacity, a drop in membrane potential and an increased production of ROS. It is possible that depleting cells of Drp1 may directly or indirectly affect mtDNA replication, distribution or segregation. In support of this hypothesis we find that mtDNA, in addition to being depleted, accumulates in discreet sections of the mitochondrial tubules and that mtDNA nucleoids on average contain more mtDNA molecules in D1 cells compared to Ctrl (Figure 4). However, it is unlikely that Drp1 is directly involved in aspects of mtDNA maintenance since it has been shown not to colocalise with mtDNA nucleoids in HeLa cells [36]. Furthermore, the levels of TFAM and mtSSB, proteins involved in mtDNA maintenance, were unchanged in D1 compared to Ctrl cells (data not shown). Nevertheless, we cannot exclude that depleting cells of Drp1 may affect the function or level of other proteins involved in mtDNA maintenance, such as the DNA polymerase PLOG, or indirectly alter mtDNA distribution by preventing fission of the mitochondrial tubule.In this study we show that the mitochondrial dysfunction resulting from the inhibition of mitochondrial fission leads to cell proliferation arrest and autophagy. Both these events are likely to be due to the drop in ATP levels in Drp1-depleted cells. Several studies have shown that inhibiting mitochondrial function, either by decreasing mitochondrial protein synthesis or by inhibiting respiration, led to ATP depletion and growth arrest [37]–[39]. Furthermore, we have found that treating HeLa cells with oligomycin or the mitochondrial uncoupler CCCP leads to an arrest in the cell cycle (data not shown).Autophagy is widely associated with conditions of nutrient starvation [22] and thus the increase in autophagy in Drp1-depleted cells possibly occurs in an effort to refuel the cellular ATP production. Although autophagy is known to remove damaged mitochondria [22], [40], we find that autophagy occurring in D1 cells does not primarily target mitochondria (Fig. 3A). The only instance in which there is co-localisation between the autophagic and mitochondrial markers in Drp1-depleted cells is when the mitochondria are small and round (arrowheads Fig. 3A). This is in agreement with results showing that Drp1-overexpression (resulting in fragmentation of the mitochondrial network) promoted disappearance of mitochondria during apoptosis, while overexpression of dominant-negative Drp1K38A prevented this elimination of the organelle [41], [42]. Therefore, although most of the long tubular mitochondria in Drp1-depleted cells are likely to be damaged, these organelles are too large to be eliminated by mitophagy and it is only when small sections of the mitochondrial tubules separate that they can be targeted for autophagic degradation.In conclusion, we have shown that an active mitochondrial fission machinery is required for maintenance of mitochondrial function and for preservation of its genome.Materials and MethodsMaterialsMost chemical compounds were purchased from Sigma-Aldrich. JC1, TMRE and DCFDA were from Molecular Probes (Invitrogen). The following antibodies were used: LDH-A (Sigma), LC3 (MBL), GAPDH (6C5, Abcam), Drp1 (DLP1, Transduction Laboratories), BrdU and cytochrome c (BD Pharmingen), DNPH and DNA (Chemicon), VDAC1 (Calbiochem), TOM20 (Santa-Cruz).Cell Culture and transfectionsHeLa CCL-2 cells (purchased from the European Collection of Cell Cultures) and 293T cells were cultured in high-glucose Dulbecco's minimal essential medium with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM glutamine and maintained in 5% CO2 at 37°C. For transient transfections cells were plated in culture dishes 45 min before transfection and transfected using a calcium phosphate coprecipitation method [43]. At 24 h after transfection the cells were washed once with Tris-buffered saline (TBS) and grown in fresh medium supplemented with 3 µg/ml puromycin (Calbiochem) for 24 h to select for the transfected cells. The cultures were then washed with phosphate-buffered saline (PBS) and incubated in fresh growth medium until the start of the experiment.The mitochondrial catalase construct was kindly provided by Dr. Peter Rabinovitch (University of Washington, Seattle, USA).RNA interference and RetrovirusesDown-regulation of Drp1 in HeLa was achieved by RNA interference using a vector-based shRNA approach [21]. The target sequences were 5′-GCAGAAGAATGGGGTAAAT-3′ for D1 (nucleotides [nt] 330 to 349; accession no. NM_012063, following advice from A. M. Van der Bliek, David Geffen School of Medicine, UCLA), 5′-GGATATTGAGCTTCAAATCA-3′ for D2 (nt 552 to 571; accession no. NM_012063). The specificity of each sequence was confirmed by BLAST searches. The oligonucleotides corresponding to these sequences were cloned into pSUPER-RETRO mammalian expression vector (kindly provided by Rewen Agami, The Netherlands Cancer Institute) as previously described [21]. To control for the potential side effects of transfecting cells with the pSUPER-RETRO vectors and expressing shRNAs, all Ctrl cells were transfected with firefly luciferase-targeted shRNA-expressing pSUPER-RETRO vector (sequence 5′-CGTACGCGGAATACTTCG A-3′) as described previously [44].The production of vesicular stomatitis virus G, pseudotyped retroviral particles encapsulating the respective pSUPER-RETRO vectors was performed as previously described [18]. Briefly, 293T cells were transfected with the pSUPER-RETRO shRNA, pCMVgag/pol and pMD2G vectors. 48 h after transfection, the culture medium containing the viral particles was collected and centrifuged at 1,000×g for 10 min at 4°C, filtered through a 0.45 µm filter, and stored at −80°C. HeLa cells were transduced by incubating actively growing cultures with the viral supernatants for 16 to 24 h.Immunoblotting and immunocytochemistryFor immunoblotting, cells were resuspended in lysis buffer: 10 mM HEPES, 300 mM KCl, 5 mM MgCl2, 1 mM EGTA, 1% Triton X-100 (vol/vol), 0.1% (wt/vol) sodium dodecyl sulfate (SDS), pH 7.4, supplemented with 1× proteinase inhibitor mixture (Roche). Lysates were spun at 2,000×g, and the protein concentration was determined by Bradford assay (Bio-Rad). Equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Schleicher & Schuell), immunoblotted with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and developed via enhanced chemiluminescence.Mitochondrial protein oxidation was determined in lysates of isolated mitochondria using the OxyBlot™ Protein Oxidation Detection Kit (Chemicon) according to the manufacturer's instruction. The anti-DNPH immunoblot was quantified and normalized to the loading control (VDAC) using ImageJ version 1.40 g.Immunocytochemistry was performed as follows: cells grown on glass coverslips were fixed in 4% paraformaldehyde diluted in growth medium for 20 min at room temperature (RT) followed by PBS washes. The cells were then permeabilized with 0.1% Triton X-100 in PBS and blocked at RT in PBS containing 0.1% Triton X-100 and 5% normal goat serum. The coverslips were then incubated with primary antibodies diluted in blocking buffer for 2 h at RT (or overnight at 4°C) followed by washes in permeabilization buffer. Immunoreactive proteins were visualized by incubating the cells with fluorescein isothiocyanate (FITC) or Texas Red-coupled mouse or rabbit secondary antibodies (Vector Laboratories) in permeabilization buffer for 1 h at RT, followed by washes in permeabilization buffer. Coverslips were mounted in a DABCO solution (2.4% DABCO-52% glycerol in PBS; pH 7.2). Fluorescent images were visualized using a Zeiss Axiovert 135TV apparatus or an Olympus IX70 Deltavision Microscope. Images were captured using a charge-coupled-device camera and processed using Adobe Photoshop CS 8.0. Quantification of the integrated fluorescent intensity of individual mtDNA nucleoids was performed on maximal projection images of 9 D1 cells and 10 Ctrl cells using MetaMorph version 6.3r7.Immunocytochemistry to quantify cell proliferation by BrdU incorporation was performed as previously described [26] on HeLa cells 120 hrs after transfection that had been incubated in medium containing 25 µM BrdU for 18 hrs. Briefly, coverslips were permeabilised as outlined above and DNA was denatured by incubating the coverslips in 2N HCl for 30 min at 37°C. The coverslips were washed extensively with 0.1 M Sodium Borate pH 8.5 and immunostained as above using anti-BrdU as the primary anitibody.ROS, ΔΨm, ATP and oxygen consumption measurementsThe intracellular level of ROS and ΔΨm of shRNA transfected cells was measured by flowcytometry of live cells stained with 40 µM Carboxy-H2DCFDA or 0.5 µM carbocyanine dye 5,5′,6,6′,-tetrachloro-1,1,3,3′tetraethylbenzimidazolylcarbocyanine iodide (JC-1) or tetramethylrhodamine ethyl ester (TMRE). Briefly, adherent cells in culture (for H2DCFDA) or cells in suspension after trypsinisation (for JC1 and TMRE) were incubated with medium (or PBS containing 0.15 g/L CaCl2, 1 g/L glucose in the case of H2DCFDA) containing the respective dyes for 15 min (for TMRE) to 1 hr (for H2DCFDA and JC1). JC1 and TMRE stained cells were then analysed by flowcytometry in growth media and H2DCFDA stained cells were washed and collected by trypsinisation PBS containing 0.15 g/L CaCl2, 1 g/L glucose before FACS analysis. Of note in order to normalise the intensity of TMRE fluorescence for the volume of mitochondria, the latter was divided by the intensity of TMRE after addition of 100 µM for the mitochondrial uncoupler CCCP.Cellular ATP levels were determined using the ATP determination kit (Molecular probes, Invitrogen) according to the manufacturer's instructions. Briefly, HeLa cells transfected for 96 hrs were collected by trypsinisation, counted and lysed (on ice of 10 min) at 105 cells per 100 ul of PLB (Promega). The lysates were spun at 12,000×g at 4°C for 5 min to pellet cell debris. The protein concentration of the cleared lysates was determined as before and triplicates of 3 µg of lysates were used for ATP determination with a D-luciferin/firefly luceferase reaction mix and luminescence was measured on a CHAMELEON™ multi-plate reader and compared to a freshly prepared ATP standard curve.Oxygen consumption measurements were made on live non-permeabilised retrovirally infected cells in growth medium (supplemented with 10 mM Hepes, pH 7.5) using a Gilson oxygraph equipped with a Clark electrode (Gilson Medical Electronic, Middleton, WI). Oxymetry on isolated organelles was performed on mitochondria isolated from retrovirally infected cells. Briefly, the cells were scraped in medium, washed once in cold PBS, and resuspended in MB (210 mM mannitol, 70 mM sucrose, 10 mM HEPES, pH 7.5, 1 mM EDTA) supplemented with protease inhibitors (Roche). Cells were broken by 15 strokes in a 2 ml glass homogeniser (Kontes Glass Co) on ice and the suspension was centrifuged at 500×g at 4°C for 5 min. The supernatant was kept, and the pellet was resuspended in homogenization buffer and homogenised as above. The suspension was spun as above and the supernatants were pooled and centrifuged for 5 min at 1,500×g at 4°C to pellet the remaining nuclei and unbroken cells. The supernatant was further centrifuged for 5 min at 10,000×g at 4°C to pellet the mitochondria. The supernatants were discarded, and the mitochondria were washed with homogenization buffer. The quantity of protein was determined as before and 100 µg of the mitochondrial fraction was resuspended in respiration buffer (210 mM Mannitol, 70 mM Sucrose, 1 mM EDTA, 4 mM Na2HPO4, 5 mM MgCl2) to determine the rate of oxygen consumption of the isolated organelles using the above Clark electrode system. State IV respiration, corresponding to the rate of uncoupled respiration, was assessed in the presence of 5 mM Succinate and the State III respiratory rate (corresponding to coupled respiration) was obtained after addition of 0.5 mM ADP to the 5 mM Succinate. The percentage of respiratory uncoupling was obtained by dividing oxygen consumption in the presence of 5 mM Succinate (State IV) by that after addition of 0.5 mM ADP (State III).Flow cytometry was performed using a Becton Dickinson FACS Track flow cytometer with CellQuest software 3.3. FL1 and FL2 corresponds the 530/30 BP and 585/42 BP emission filters respectively.Quantification of mtDNA by Q-PCRQuantification of the mtDNA was performed as previously described by Legros and colleagues [26]. Briefly, 100,000 cells were resuspended in 100 µl extraction solution (0.2 mg/ml proteinase K, 0.2% SDS and 5 mM EDTA in PBS) and incubated at 50°C for 3 h. Total DNA was then precipitated by addition of 10 µl of 3 M sodium acetate (pH 5.2), 110 µl isopropanol and incubation for 20 minutes on ice before centrifugation at 12,000 rpm at 4°C. The DNA-pellet was washed once with cold 70% ethanol, air dried for 15 min and resuspended in 100 µl TE buffer at 4°C overnight. Realtime PCR amplification was performed on 10 ng of total DNA using a iCycler (Bio Rad) and iQ SYBR Green Supermix (BioRad) following the manufacturer's instructions. A 211 bp fragment of the mtDNA 12S RNA gene was amplified between nucleotide 1095 and nucleotide 1305 (Forward primer: 5′ GCTCGCCAGAACACTACGAG 3′, reverse primer: 5′ CAGGGTTTGCTGAAGATGGCG 3′). Elongation translation factor 1 gene (EEF1A1) was used as an endogenous reference across all experimental conditions (Forward primer: 5′ GGATTGCCACACGGCTCACATT 3′, reverse primer: 5′ GGTGGATAGTCTGAGAAGCTCTC 3′).Supporting InformationFigure S1Depleting HeLa cells of Drp1 using the D1 or D2 construct inhibits mitochondrial fission A. HeLa cells were transiently transfected with the Ctrl, D1 or D2 constructs, selected with puromycin for 24 h and collected for Western blotting analysis using the indicated antibodies 96 h after transfection. B. HeLa cells transfected with the Ctrl, D1 or D2 constructs and treated as in A. were immunostained with a rabbit TOM20 antibody 96 h after transfection. The scale bar corresponds to 15 µm.(2.87 MB TIF)Click here for additional data file.\n\nREFERENCES:\n1. Bereiter-HahnJVothM\n1994\nDynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria.\nMicrosc Res Tech\n27\n198\n219\n8204911\n2. 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+    "id": "PMC2532956",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2532956\nAUTHORS: C Wright, R Lakshman, P Emmett, K K Ong\n\nABSTRACT:\nBackground:The WHO 2006 Child Growth Standard is based on data from international optimally nourished breastfed infants from birth to age 5 years.Objective:To assess the potential effect of its use on weight and growth monitoring of UK children.Participants:Full-term members of two population-based UK birth cohorts: the Children in Focus sub-cohort of the Avon Longitudinal Study of Parents and Children (ALSPAC) (n = 1335) and the Gateshead Millennium Baby Study (GMS; n = 923).Design:Growth data from birth to 5 years were converted into z-scores relative to the WHO 2006 standard.Results:Compared with the WHO standard, both UK cohorts had higher birth weights (mean z-scores: GMS, 0.17; ALSPAC, 0.34) and ALSPAC had higher birth lengths. After birth, length showed a good fit at all ages. By 2–4 months, both cohorts were similar in weight to the WHO median (mean WHO weight z-score at 4 months: GMS, 0.01; ALSPAC, −0.07), but thereafter the UK cohorts were heavier (mean WHO weight z-score at 12 months: GMS, 0.57; ALSPAC, 0.65). At age 12 months, the risk of being classified as underweight (weight <2nd centile) was considerably lower according to the WHO standard than by the UK 1990 Growth Reference (RR = 0.15, 95% CI = 0.07 to 0.32), and the risk of being classified as obese at 4–5 years (body mass index >98th centile) was slightly increased (RR = 1.35, 95% CI = 1.02 to 1.78).Conclusions:Adoption of the WHO 2006 Growth Charts would set a markedly lower standard of weight gain beyond the age of 4 months for UK infants and could support efforts to avoid future childhood obesity. However, the WHO standard is not representative of size at birth in the UK.\n\nBODY:\nThe World Health Organisation (WHO) Child Growth Standard for infants and children up to the age of 5 years was published in April 2006. It is based on the growth of healthy breastfed children in optimal conditions between 1997 and 2003 from six different countries: Brazil, Ghana, India, Norway, Oman and USA.1 2 The WHO Multicentre Growth Reference Study (MGRS) collected data for ∼8500 children who were exclusively breast fed for the first 4 months and were living in a well-supported health environment. In consequence, the WHO aims to provide for the first time a standard on “how children should grow”, rather than a traditional growth reference that describes “how children are growing”.There is an understandable enthusiasm for the idea of adopting these charts in the UK, but before doing so it is important to assess how well UK children match, or diverge from, the new charts, in order to understand the implications for growth monitoring and clinical care. We have explored this question using data from two representative UK birth cohorts.METHODSThe two datasets used were from the Gateshead Millennium Baby Study (GMS) and the Children in Focus sub-sample of the Avon Longitudinal Study of Parents and Children (ALSPAC), which between them provide detailed growth data spanning the entire period of the new charts. GMS is a prospective population-based cohort study of feeding and growth in infancy comprising 1029 babies born between June 1999 and May 2000 in Gateshead, an urban borough in the North of England. For this analysis, data from 923 full-term infants were used.3 Birth weight was retrieved from the maternity record, and weights at 12 days, 6–8 weeks, 4 months and 12 months were obtained from the Personal Child Health Records as well as height and weight at school entry.3 Half were breast fed at birth, but only 10% continued breast feeding beyond 4 months.The ALSPAC Children in Focus sub-cohort includes 1335 full-term infants born in Avon, south-west England, between June and December 1992. Weight and length/height measurements were collected at research clinics at birth, 4 months, 8 months, 12 months, 18 months, 24 months and 5 years.4 Just less than half (46%) were breast fed at age four months (including up to one formula feed per day).For each child, age- and sex-adjusted z-scores for weight, length (height at >2 years old) and body mass index (BMI) were calculated using exact ages at measurement by comparison with both the WHO 2006 and the UK 1990 growth data using software provided respectively by the WHO and the Child Growth Foundation (London, UK). Conditional weight gain was calculated to account for regression to the mean.3 Poor infant weight gain was defined as a change in weight SD score <−1.33 SD, which is equivalent to downward crossing through two major centile lines on each growth chart.Both studies received appropriate ethics committee approval and obtained informed written consent from each participant.RESULTSComparisons with UK 1990Both cohorts showed a reasonably good fit with the UK 1990 reference during the first year of life, as indicated by mean weight and length z-scores close to zero (table 1 and fig 1). The only exception was a transient decline in weight z-score in GMS at age 12 days, which may be expected, as the UK 1990 reference makes no allowance for the physiological neonatal weight loss. By age 4–5 years, weight and BMI z-scores in both cohorts were higher than the UK 1990 average.Figure 1Mean z-scores for weight from birth to 24 months and at 4–5 years, according to WHO 2006 Growth Standard (WHO 2006) or the British 1990 Growth Reference (UK 1990) for the Gateshead Millennium Baby Study (GMS) and the Children in Focus sub-cohort of the Avon Longitudinal Study of Parents and Children (ALSPAC). Dotted lines in each panel indicate the time periods with less density of measurements.Table 1z-Scores for length/height, weight and body mass index (BMI) from birth according to the WHO 2006 Growth Standard (WHO) or the British 1990 Growth Reference (UK1990) in the ALSPAC and GMS cohortsAgeNumbersLength/height SDSWeight SDSBMI SDSWHOUK1990WHOUK1990WHOUK1990ALSPACGMSALSPACGMSALSPACGMSALSPACGMSALSPACGMSALSPACGMSALSPACGMSBirth13359230.65 (1.04)–0.04 (1.00)–0.34 (1.01)0.17 (1.07)−0.03 (1.04)−0.20 (1.09)0.00 (0.98)–0.17 (1.00)–12 days–806–––––−0.07 (1.00)–−0.51 (1.03)––––6–8 weeks–788–––––−0.17 (0.93)–−0.02 (0.99)––––4 months943796−0.03 (0.91)–−0.05 (0.90)–−0.07 (0.90)0.01 (0.96)−0.12 (0.97)0.05 (1.03)−0.07 (0.94)–−0.12 (1.29)–8 months12316010.15 (0.96)–0.08 (0.96)–0.46 (0.97)0.49 (0.94)0.10 (1.08)0.22 (1.04)0.50 (0.96)–0.09 (1.06)–1 year11647740.09 (0.95)–0.09 (0.94)–0.65 (0.93)0.57 (0.94)0.18 (1.05)0.14 (1.05)0.81 (0.89)–0.19 (1.01)–1.5 years1088–−0.09 (0.97)–0.02 (0.96)–0.51 (0.91)–0.08 (1.04)–0.81 (0.89)–0.06 (1.03)–2 years977–−0.14 (0.93)–−0.11 (0.93)–0.40 (0.92)–0.15 (1.04)–0.66 (0.93)–0.26 (1.03)–4–5 years963395*−0.11 (0.91)−0.16 (0.93)0.08 (0.94)−0.01 (0.97)0.35 (0.90)0.49 (0.96)0.31 (1.00)0.45 (1.03)0.63 (0.92)0.87 (0.99)0.42 (0.99)0.66 (0.99)Values are mean (SD).*281 GMS children aged >5 years at school entry measurement were excluded as they could not be compared with WHO.ALSPAC, Children in Focus sub-cohort of Avon Longitudinal Study of Parents and Children; GMS, Gateshead Millennium Baby Study.Comparisons with WHO 2006UK children had relatively high mean z-scores for birth weight and birth length compared with the WHO 2006 standard (table 1). After birth, z-scores for weight in the GMS children rapidly declined towards the WHO median by age 2 weeks, and in both cohorts weight showed a good fit up to 4 months (fig 1). Length and height in both cohorts showed a good fit at all ages after birth (table 1).Between 4 months and 1 year, compared with the WHO standard, both cohorts showed a rapid rise in mean weight z-scores. After 1 year, the mean z-scores as assessed by the different growth reference data started to converge (table 1, fig 1).By the WHO 2006 standard, infants were considerably less likely to be classified as underweight (weight <2nd centile; relative risk at 1 year = 0.15; 95% CI = 0.07 to 0.32) or having poor weight gain (downward-crossing through weight centiles) over the first year, compared with the UK 1990 Reference (table 2). Conversely the proportion of children classified as obese (BMI >98th centile) at age 4–5 years was slightly higher according to the WHO 2006 standard (relative risk = 1.35; 95% CI = 1.02 to 1.78; table 2).Table 2Percentages of children classified as underweight, poor infant weight gain, or obese according to the WHO Growth Standard (WHO) and the British 1990 Growth Reference (UK1990)ALSPACGMSCombined:RR (95% CI)WHOUK1990WHOUK1990Underweight    6–8 weeks––3.62.91.12 (0.87 to 1.43)    4 months2.02.42.12.40.86 (0.55 to 1.33)    8 months0.72.60.71.80.30 (0.17 to 0.56)    1 year0.32.40.42.50.15 (0.07 to 0.32)    1.5 years0.62.9––0.22 (0.10 to 0.48)Poor infant weight gain    Birth to 1 year1.77.11.65.40.24 (0.16 to 0.36)    6–8 weeks to 1 year––0.45.40.08 (0.03 to 0.24)Obese (%)    1 year8.72.7––3.26 (2.21 to 4.83)    1.5 years8.02.6––3.11 (2.06 to 4.71)    2 years7.54.3––1.74 (1.20 to 2.51)    4–5 years7.25.010.18.41.35 (1.02 to 1.78)ALSPAC, Children in Focus sub-cohort of Avon Longitudinal Study of Parents and Children; GMS, Gateshead Millennium Baby Study; RR, relative risk for each outcome using the WHO standard, compared with the UK 1990 reference; Underweight, weight <2nd centile; Poor infant weight gain, conditional weight gain <−1.33 SD, equivalent to downward crossing through two major centile lines on each growth chart; Obese, body mass index >98th centile.DISCUSSIONIn summary, adoption of the new WHO growth charts for UK children up to age 5 years would have a significant impact on the interpretation of their weight gain and growth. However, the effects are complex and appear to differ at various ages. The marked reduction in numbers of infants who would be classified as underweight or growth faltering beyond age 4 months is an expected consequence of the WHO’s decision to have the breastfed child as the normative model. However, UK infants would also be classified as being larger at birth, but not at 2–4 months, and would result in a complex pattern of weight centile changes over the first year for the average UK child (fig 1).This analysis is based on data from two large representative UK birth cohorts, which between them allow comparison with the WHO charts at a wide range of ages. GMS provides detailed weight data early in infancy, and the ALSPAC provides both weight and height/length from infancy through to the pre-school years. At times of overlap, the two cohorts showed very close similarity in weights and heights, and, at least in infancy, they are also broadly similar to the UK 1990 reference. The gradual increase in weight z-scores by 4–5 years of age compared with the UK 1990 has been previously reported in ALSPAC and probably reflects the secular changes in UK children.5 We are therefore confident that our findings in these two cohorts may be extrapolated to contemporary UK children.The WHO 2006 Child Growth Standard embodies a number of novel and admirable principles, with the aim of promoting optimal infant and childhood growth. Firstly, the international MGRS source data indicated for the first time that population differences in growth are avoidable, given optimum nutrition and living conditions.6 Secondly, the WHO has clearly placed the breastfed child as the norm for growth and development. Conditions of inclusion in the longitudinal component of the MGRS analysis were exclusive or predominant breast feeding up to age 4 months and partial breast feeding to at least 12 months. In consequence, the WHO feels able to publish a standard for optimal growth, rather than simply a description of current prevailing growth norms (a “reference”), which may not reflect ideal growth patterns.What is already known on this topicThe WHO published new growth charts in April 2006 based on infants of non-smoking, breastfeeding mothers living in optimal conditions in six countries.The WHO proposed that these set a standard for normal growth in infancy applicable throughout the world.What this study addsAt birth, UK children are longer and heavier than the WHO standard,After birth, the length of UK children matches the WHO standard closely.Use of the WHO standard would lead to far fewer UK children being classified as underweight or weight faltering in the first year, but more would be classified as overweight in the pre-school years.The WHO 2006 Growth Charts would set a lower standard of weight gain for UK infants.However, our findings, particularly during the first 2 months, suggest that these standards may not be simply transferable to the UK. On the WHO chart, UK infants would appear larger than average at birth and then cross approximately half a centile space downwards in the first few weeks of life. The explanation for this may be that, although postnatal nutrition in the WHO MGRS cohort was optimal, intrauterine growth appeared to have been constrained, as size at birth was generally smaller than in the UK. In the MGRS constituent datasets, whereas mean birth weights in Norway and USA (3.5–3.6 kg) were similar to that in the UK, the populations from several other countries showed markedly lower mean birth weights (3.1 kg in India, and 3.2 kg in Oman), and this appears to correlate with differences in maternal size.7The UK 1990 and other existing national growth charts do not allow for the rapid weight loss and recovery that normally occurs in the first 2 weeks of life.8 This is reflected in fig 1 by a transient dip in the GMS cohort UK 1990 weight SD scores at age 12 days, which probably corrected itself well before their next measurement at age 6 weeks. In contrast, the WHO standard does allow for normal neonatal weight loss.2 Therefore, the apparent downward shift in weight centile of UK children on the WHO chart after birth (fig 1) is not simply a transient physiological weight loss, but rather suggests that individual babies with low birth weight in the international MGRS birth cohort showed rapid catch-up growth after birth, even within the first 2 weeks.Beyond the first 2–4 months, use of the WHO standard would make it much less likely for UK children to be classified as underweight or growth faltering. Recent work has revealed that mild degrees of weight faltering are unlikely to be associated with major social or medical disorders,3 and concerns have been expressed that unnecessary parental anxiety may be caused by over-diagnosis.9 A change to a new standard, with a more stringent and thus more specific lower threshold, may therefore be timely.In contrast with underweight, adoption of the WHO growth chart would make UK infants and toddlers more likely to be classified as overweight or obese. There is a growing body of evidence that a higher plane of growth during infancy is associated with increased risk of obesity in children and adults.10 11 Although it is not at all clear whether intervention in infancy can have a useful impact on later obesity, presenting the model of slower weight gain during later infancy prescribed by the WHO standard may be beneficial to the long-term health of these children.The birth weight section of the WHO chart presents other difficulties, as there is no preterm element, which is a well-used feature of UK charts. These two issues taken together suggest that it may not be desirable for the UK to adopt the birth weight section of the WHO chart, beginning its use instead after the first 2 weeks.In conclusion, the WHO 2006 Growth Standard places the breastfed child as the norm for growth. Its use would greatly reduce the numbers of UK infants classified as underweight and support efforts to avoid excess weight gain in infancy. However, the WHO 2006 Growth Standard is not representative of size at birth in the UK. In view of the resulting complex weight centile changes in the first few weeks of life, the potential confusion about feeding that this might raise with mothers, and also the absence of a preterm element to the WHO charts, the Department of Health Scientific Advisory Committee on Nutrition and the Royal College of Paediatrics and Child Health have recently jointly recommended that the WHO 2006 Growth Standard is appropriate for use in the UK children, but only from age 2 weeks.12 For birth weight, the UK 1990 reference would continue. The consequences of these recommendations for monitoring of infant weight gain in the UK are likely to be widespread and will need careful and coordinated consideration.\n\nREFERENCES:\n1. de OnisMGarzaCVictoraCG  The WHO Multicentre Growth Reference Study: planning, study design, and methodology. Food Nutr Bull 2004;25:S15–2615069916\n2. WHO Child Growth Standards based on length/height, weight and age Acta Paediatr Suppl 2006;450:76–8516817681\n3. WrightCMParkinsonKNDrewettRF The influence of maternal socioeconomic and emotional factors on infant weight gain and weight faltering (failure to thrive): data from a prospective birth cohort. Arch Dis Child 2006;91:312–1716397011\n4. OngKKAhmedMLEmmettPM  Association between postnatal catch-up growth and obesity in childhood: prospective cohort study. BMJ 2000;320:967–7110753147\n5. ReillyJDorostyAEmmettP Prevalence of overweight and obesity in British children: cohort study. BMJ 1999;319:103910521196\n6. Assessment of differences in linear growth among populations in the WHO Multicentre Growth Reference Study Acta Paediatr Suppl 2006;450:56–6516817679\n7. Enrolment and baseline characteristics in the WHO Multicentre Growth Reference Study Acta Paediatr Suppl 2006;450:7–1516817674\n8. WrightCMParkinsonKN Postnatal weight loss in term infants: what is normal and do growth charts allow for it? Arch Dis Child Fetal Neonatal Ed 2004;89:F254–715102731\n9. SpencerNJ Failure to think about failure to thrive. Arch Dis Child 2007;92:95–617264277\n10. BairdJFisherDLucasP  Being big or growing fast: systematic review of size and growth in infancy and later obesity. BMJ 2005;331:92916227306\n11. OngKKLoosRJ Rapid infancy weight gain and subsequent obesity: systematic reviews and hopeful suggestions. Acta Paediatr 2006;95:904–816882560\n12. Department of Health Application of the WHO Growth Standards in the UK. http://www.sacn.gov.uk/ (accessed 21 Nov 2007)"
+}

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+{
+    "id": "PMC2533005",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533005\nAUTHORS: Hao-Tsai Cheng, Chi-Liang Cheng, Cheng-Hui Lin, Jui-Hsiang Tang, Yin-Yi Chu, Nai-Jen Liu, Pang-Chi Chen\n\nABSTRACT:\nBackgroundThe ingestion of caustic substances induces an extensive spectrum of injuries to the aerodigestive tract which include extensive necrosis and perforation of the esophagus and stomach. The gold standard of safely assessing depth, extent of injury, and appropriate therapeutic regimen is esophagogastroduodenoscopy (EGD). The objective of this study was to report our clinical experience and to evaluate the role of a 6-point EGD classification system of injury in predicting outcomes in adult patients diagnosed with caustic agent ingestion.MethodsThe study was a retrospective medical chart review from 273 patients admitted to the Chang Gung Memorial Hospital in Tao-Yuan, Taiwan between June 1999 and July 2006 for treatment of caustic ingestion. The patients underwent EGD within 24 hours of admission and mucosal damage was graded using Zagar's modified endoscopic classification scheme. After treatment, patients were followed in the outpatient clinic for a minimum of 6 months.ResultsA total of 273 patients were included for analysis. Grade 3b injury was the most common caustic injury (n = 82, 30.03%), followed by grade 2b injuries (n = 62, 22.71%). Stricture was the most common complication (n = 66, 24.18%), followed by aspiration pneumonia (n = 31, 11.36%), and respiratory failure (n = 21, 7.69%). Compared to grade 3a mucosal injury, grade 3b mucosal injuries were at greater risk of prolonged hospital stay (odds ratio [OR]: 2.44; 95% confidence interval [CI]: 1.25–4.80), ICU admission (OR: 10.82; 95% CI: 2.05–200.39), and gastrointestinal (OR: 4.15; 95% CI: 1.55–13.29) and systemic complications (OR: 4.07; 95% CI: 1.81–14.07).ConclusionIn patients with caustic ingestion, EGD should be performed within 12 to 24 hours and categorized according to a 6-point scale. Patients with grade 3b burns identified on endoscopy have high rates of morbidity. The 6-point scale is useful for predicting immediate and long-term complications, and guiding appropriate therapy.\n\nBODY:\nBackgroundThe ingestion of caustic substances induces a wide range of injuries to the gastrointestinal tract, which can be mild or fatal, or lead to chronic disease [1]. Caustic ingestion in children is usually accidental ingestion [2], while ingestion by adults is often due to suicidal intent, and injuries tend to be more severe [3].Caustic agents with a pH level <2 or >12 rapidly penetrate layers of the esophagus resulting in necrosis-induced eschar formation in the mucosa that limits deep tissue penetration [4]. The extent of tissue destruction depends on the physical form, type, and concentration of corrosive agent, premorbid state of the tissue, contact duration, and amount of substance ingested. Esophageal mucosa is thought to be more resistant to acidic than alkaline substances, as alkaline liquids are often highly viscous and thus persist for a longer duration in the esophageal mucosa [5]. Liquefaction necrosis occurs and serious esophageal injury becomes inevitable once alkaline liquids penetrate deep muscle layers [6].The gold standard of safely assessing depth, extent of caustic ingestion injury, and appropriate therapeutic regimen is esophagogastroduodenoscopy (EGD). Indications, mucosal injury classification, optimal timing, and the degree of esophageal injuries that necessitate EGD in relation to treatment regimens, however, are matters of debate [4-10]. The objective of this study was to report our clinical experience and to evaluate the role of a 6-point EGD classification system of injury in predicting outcomes and guiding therapy in adult patients diagnosed with caustic agent ingestion.MethodsA retrospective chart review of 288 adult patients (>18 years of age) who were admitted to Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, for caustic ingestion between June 1999 and July 2006 was conducted. Parameters analysed were age, gender, intent of ingestion, substance ingested and amount, time to expiration, ICU admittance, length of hospital stay, complications, and the severity of mucosal injury as assessed by EGD.EGD with a standard upper GI endoscope was performed by experienced physicians within 24 hours of ingestion. Endoscopes used were Olympus GIF XQ-230, GIF Q-240X, and GIF Q-260, with diameters of 9.2 mm, 9.4 mm, and 9.2 mm, respectively (Olympus, Tokyo, Japan). Oral cavity xylocaine spray was used for anaesthesia except in 15 cases, which received ventilation support under general anaesthesia because of respiratory difficulty (n = 11) or unclear consciousness (n = 4). Gentle insufflations and retrovisual methods were performed carefully or avoided in the presence of severe stomach injury. Mucosal damage was graded using a modified endoscopic classification described by Zagar et al [11] (Table 1).Table 1Zargar's grading classification of mucosal injury caused by ingestion of caustic substancesGrade 0Normal examinationGrade 1edema and hypermia of the mucosaGrade 2aSuperficial ulceration, erosions, friability, blisters, exudates, hemorrhages, whitish membranesGrade 2bGrade 2a plus deep discrete or circumferential ulcerationsGrade 3aSmall scattered areas of multiple ulceration and areas of necrosis with brown-black or greyish discolorationGrade 3bExtensive necrosisPatients were treated with a proton pump inhibitor or H2 antagonist and were maintained without oral intake until their condition was considered stable. Patients received parenteral nutrition during this period. If infection was suspected, antibiotics (a 1st generation cepholasporin and gentamicin) were administered after blood cultures were obtained. If a patient's condition destablized or respiratory difficulty was encountered, they were transferred to the intensive care unit for further evaluation. After discharge, patients were followed in the outpatient clinic for at least 6 months. Any complications observed during follow-up were recorded. Upper GI complications included bleeding, perforation, and stricture formation. Bleeding was defined as melena, hematemasis, and/or coffee-ground vomitus. Perforation was diagnosed by the presence of free air on a plain chest radiograph. Stricture was defined as dysphagia, symptoms of regurgitation, or difficulty in swallowing with confirmation by endoscopy, esophagogram, and/or upper GI radiography. Systemic complications included renal insufficiency, liver damage, diffuse intravascular coagulation, and hemolysis. Liver damage was defined as an elevation in the serum level of alanine aminotransferase or asparatate aminotransferase greater than 3 times the upper normal limit. Renal insufficiency was defined as a plasma creatinine level of >1.4 mg/dL in the absence of other renal diseases. Criteria for disseminated intravascular coagulation and/or hemolysis were prolonged plasma coagulation time, decreased fibrinogen or antithrombin levels, and decreased platelet count.Demographic data were described by mean and standard deviations for normally distributed continuous variables, median and interquartile range for non-normally distributed continuous variables, and frequencies and percentages for categorical variables. Wald's Chi-Square tests adjusted for age obtained by generalized estimation equations were used to evaluate for overall survival and complications over grade of mucosal injury. Data subset was subsequently analyzed using logistic regression. Data were analyzed using SAS 9.0 (SAS Institute Inc, Cary, NC, US), and P < 0.05 was considered significant.ResultsA total of 273 patients consisting of 127 (47%) males and 146 females (53%) with a mean age of 43.77 ± 18.46 were included in our analysis (Table 2). One patient attempted suicide twice with different corrosive substance in a 3-month period. Fifteen patients were excluded from analysis as a result of missing data (n = 14) or endoscopy failure due to severe laryngeal edema (n = 1). Ingestion intent was primarily attributed to suicide (n = 194, 71.06%) while 28.94% (n = 79) of the cases were accidental. The amount of ingested substance ranged from 2 ml to 3000 ml and was estimated based on the history given by the patient or family member. Industrial cleaning agents containing lye or other alkaline chemical (ie, caustic soda, drain cleaners, machine cleaners, and deacidification products containing sodium hydroxide or sodium-potassium hydroxide, dishwater detergents) or caustic acids were considered caustic substances. Ingestion of industrial cleaning agents (n = 131, 47.99%) and strong acids (n = 95, 34.80%) comprised the majority of cases. Other caustic substances such as pesticides, caustic food, drugs, and other substances comprised the rest of the cases. Of these, 35.16%, 34.43%, and 30.40% were alkaline, hydrochloric acid, or unidentified acid-based substances, respectively.Table 2Patient demographic features and caustic ingestion characteristics by endoscopic grade of mucosal injuryVariablesOverallEndoscopic Grade(n = 273)012a2b3a3bP(n = 3)(n = 31)(n = 56)(n = 62)(n = 39)(n = 82)Age43.77 ± 18.4637.00 ± 27.7840.45 ± 15.5442.21 ± 18.9640.92 ± 16.7244.10 ± 17.7848.32 ± 19.940.0107*Male127 (46.52)3 (100)16 (5.93)31 (11.48)26 (9.63)16 (5.93)35 (12.96)0.1534Accidental Ingestion79 (28.94)1 (33.33)10 (32.26)19 (33.93)14 (22.58)15 (38.46)20 (24.39)0.3820Class of Substance Alkaline96 (35.16)3 (100)9 (29.03)22 (39.29)24 (38.71)13 (33.33)25 (30.49)0.5302 Acid HCL94 (34.43)0 (0.00)12 (38.71)13 (23.21)23 (37.10)11 (28.21)35 (42.68)0.1498 Other Acid83 (30.40)0 (0.00)10 (32.26)21 (37.50)15 (24.19)15 (38.46)22 (26.83)0.4086Type of Substance Industrial cleaning131 (47.99)2 (66.67)15 (48.39)26 (46.43)28 (45.16)18 (46.15)42 (51.22)0.9574 Drug3 (1.10)0 (0.00)0 (0.00)2 (3.57)1 (1.61)0 (0.00)0 (0.00)0.2862 Pesticide13 (4.76)0 (0.00)1 (3.23)5 (8.93)3 (4.84)4 (10.26)0 (0.00)0.0194* Strong acid95 (34.80)1 (33.33)11 (35.48)15 (26.79)24 (38.71)14 (35.90)30 (36.59)0.7028 Food12 (4.40)0 (0.00)2 (6.45)2 (3.57)2 (3.23)2 (5.13)4 (4.88)0.9265 Others19 (6.96)0 (0.00)2 (6.45)6 (10.71)4 (6.45)1 (2.56)6(7.32)0.7013Package Volume (ml)100 (2–3000)20 (10–100)50 (30–100)100 (35–150)100 (50–150)100 (50–120)100 (50–200)0.4423* P < 0.05 statistical significanceData presented as median (range) or N (%), endoscopic grade 0 is not included in the comparison for small contribution and possible confoundingThe results of EGD in this study showed that grade 3b injuries were the most common caustic injury (n = 82, 30.04%), followed by grade 2b injuries (n = 62, 22.71%) (Table 2). Of the 82 grade 3b patients, the esophagus was inspected in 100% of patients, the stomach was inspected in 98% (80/82), and duodenum was inspected in 84% (69/82). Severe injuries were observed in the stomach (n = 116, 42.50%), the duodenum (n = 120, 43.1%), and the esophagus (n = 71, 26.00%). Age distribution among grades of mucosal injury was significantly different (P = 0.0107, Table 2) and was subsequently used as an adjusting factor.Table 3 illustrates select variables that influenced caustic injury survival and associated complications compared with the grades of mucosal injury. Overall, the mean hospital stay was 8 days (range 0–90); hospital mortality was 6.59% (18/273); and 29 patients were admitted to the ICU. Deaths occurred from 3 days to 2 months after ingestion of the substance as a result of esophageal perforation (n = 1), tracheal perforation with active bleeding (n = 1), hematemesis with sudden apnea (n = 4), lung cancer (n = 1), or multiple organ failure (n = 11). Seventy-six patients (27.8%) developed GI complications and 20.5% (56/273) of patients developed systematic complications. Stricture formation was the most common complication observed in all patients (n = 66, 24.18%) and patients with grade 3b mucosal injury (n = 44, 53.66%), followed by aspiration pneumonia (n = 31, 11.36% vs. n = 20, 24.39%) and respiratory failure (n = 21, 7.69% vs. n = 16, 19.51%).Table 3Select parameters compared with endoscopic grade of mucosal injuryVariablesOverall (n = 273)Endoscopic Grade0 (n = 3)1 (n = 31)2a (n = 56)2b (n = 62)3a (n = 39)3b (n = 82)P†Hospital Stay (days)8 (0, 90)4 (3, 10)2 (1, 51)4.5 (0, 87)7 (2, 44)9 (0, 33)13 (0, 90)0.0147*Expired18 (6.59)0 (0.0)0 (0.0)1 (1.79)2 (3.23)1 (2.56)14 (17.07)0.0648Time to Expired (days)15.5 (3, 56)NANA11 (11, 11)16 (15, 32)16 (16, 16)15 (3, 56)--ICU Admission29 (10.62)0 (0.0)2 (6.45)2 (3.57)5 (8.06)1 (2.56)19 (23.17)0.0244*Systemic Complication56 (20.51)1 (33.33)3 (9.68)4 (7.14)11 (17.74)5 (12.82)32 (39.02)0.0111* Aspiration Pneumonia31 (11.36)1 (33.33)2 (6.45)1 (1.79)4 (6.45)3 (7.69)20 (24.39)0.0068* Respiratory Failure21 (7.69)0 (0.0)2 (6.45)1 (1.79)2 (3.23)0 (0.0)16 (19.51)0.0023* DIC10 (3.67)0 (0.0)0 (0.0)1 (1.79)1 (1.61)0 (0.0)8 (9.76)0.1524 Hepatic10 (3.67)0 (0.0)0 (0.0)1 (1.79)4 (6.45)2 (5.13)3 (3.66)0.6841 Renal7 (2.56)0 (0.0)0 (0.0)1 (1.79)1 (1.61)0 (0.0)5 (6.10)0.4300Gastrointestinal Complication76 (27.84)0 (0.0)0 (0.0)4 (7.14)9 (14.52)12 (30.77)51 (62.20)<0.0001* Stricture66 (24.18)0 (0.0)0 (0.0)2 (3.57)9 (14.52)11 (28.21)44 (53.66)<0.0001* Bleeding13 (4.76)0 (0.0)0 (0.0)2 (3.57)1 (1.61)2 (5.13)8 (9.76)0.3656 Perforation6 (2.20)0 (0.0)0 (0.0)1 (1.79)0 (0.0)0 (0.0)5 (6.10)0.2306 Fistula2 (0.73)0 (0.0)0 (0.0)1 (1.79)0 (0.0)0 (0.0)1 (1.22)0.6302†Wald's Chi-Square test*P < 0.05 statistical significanceData presented as median (range) or number (%). Endoscopic grade 0 is not included in the comparison secondary to small contribution.Stricture formation typically occurred 2 weeks after caustic ingestion. Management of the 66 patients with a stricture included gastrojejunostomy (n = 24), dilation with endoscope (n = 21), medical treatment (n = 10), esophagectomy (n = 5), jejunostomy (n = 4), esophago-colonic bypass (n = 1), and nasogastric feeding due to old CVA (n = 1). Of the 21 patients dilated endoscopically, 11 patients required subsequent surgery due to perforation (n = 3, one in the esophagus, two in the pyloric area) and failure of dilation (n = 8). Gastrojejunostomy were performed due to gastric outlet obstruction or EC junction stricture. The time of operation was determined by the patient's symptoms and signs. Fifty-one patients received surgery due to perforation (n = 6) and stricture (n = 34), and 11 patients required surgery after endoscopic dilation. Four deaths (in 51 patients who required surgery) were due to multiple organ failure, sepsis, or hematemesis.The majority of complications were observed in patients with grade 3b burns, and these were more likely to result in prolonged hospital stay (n = 13), death (n = 14), and ICU admission (n = 19). Statistical significance was observed in duration of hospital stay, ICU admittance, systematic complications, aspiratory pneumonia, respiratory failure, GI complications, and GI stricture among patients with different grades of mucosal injury (all P < 0.05; Table 3).Table 4 shows the odds ratio of endoscopic grades 2a versus 2b, and 3a versus 3b among selected variables. Grade 2b mucosal injuries were 2.5 times more likely to result in longer hospital stay (95% CI: 1.32–4.87, P < 0.05) than 2a. Other variables analysed did not show a statistically significant difference. Grade 3b mucosal injuries were 2.4 times more likely to result in longer hospital stay (95% CI: 1.25–4.80, P < 0.05), and 10.8 times more likely to be admitted to the ICU (95% CI: 2.05–200.39, P < 0.05), than grade 3a injuries. Additionally, patients with grade 3b burn injuries were 4.1 times more likely to develop systematic complications (95% CI: 1.55–13.29, P < 0.05), 4.07 times more likely to develop GI complications (95% CI: 1.81–9.69, P < 0.05) and 3.34 times more likely to develop stricture (95% CI: 1.47–8.09, P < 0.05) than those with grade 3a burns.Table 4Select parameters and odds ratio of endoscopic grade 2a versus 2b, 3a versus 3bVariablesEndoscopic Grading 2a vs. 2b OR (95% CI)PEndoscopic Grading 3a vs.3b OR (95% CI)PMedian Hospital Stay (days)2.52 (1.32, 4.87)0.0052*2.44 (1.25, 4.80)0.0102*Expired (days)1.88 (0.17, 41.13)0.61247.17 (1.32, 133.49)0.0640ICU Admission2.37 (0.49, 17.02)0.315910.82 (2.05, 200.39)0.0241*Systemic Complication Overall2.89 (0.92, 11.03)0.08714.15 (1.55, 13.29)0.0083* Aspiratory Pneumonia4.12 (0.57, 83.15)0.21633.58 (1.10, 16.22)0.0553 Respiratory Failure1.77 (0.16, 38.89)0.6453---- DIC0.91 (0.04, 23.27)0.9443---- Hepatic3.74 (0.53, 74.44)0.24480.82 (0.13, 6.48)0.8280 Renal0.95 (0.04, 24.54)0.9710----Gastrointestinal Complication Overall2.10 (0.66, 8.47)0.21834.07 (1.81, 9.69)0.0010* Stricture4.56 (1.11, 30.86)0.05963.34 (1.47, 8.09)0.0053* Bleeding0.41 (0.02, 4.50)0.47942.05 (0.48, 14.07)0.3818 Perforation-------- Fistula--------* P < 0.05 indicates statistical significance.Table 5 shows select variables compared with acid and alkali ingestion. Statistical significance was observed in duration of hospital stay only (P = 0.0419).Table 5Select parameters compared with alkali and acid ingestion groupsVariablesAlkali (n = 96)AcidP†HCL (n = 94)Other acid (n = 83)Hospital Stay (days)8 (4,16)8 (3,14)7 (3,13)0.0419*Expired6 (6.25)18 (6.59)7(8.43)0.8073Time to Expired (days)15 (11–16)15.5 (11–23)22 (13–32)--ICU Admission12 (12.50)29 (10.62)8 (9.64)0.5074Systemic Complication27 (28.13)56 (20.51)13 (15.66)0.0525 Aspiration Pneumonia13 (13.54)31 (11.36)8 (9.64)0.5188 Respiratory Failure10 (10.42)21 (7.69)6 (7.23)0.1874 DIC4 (4.17)10 (3.66)4 (4.82)0.4754 Hepatic7 (7.29)10 (3.66)1 (1.20)0.0511 Renal3 (3.13)7 (2.56)2 (2.41)0.6618GI Complication36 (37.50)76 (27.84)15 (18.07)0.0818 Stricture31 (32.29)66 (27.84)12 (14.46)0.1855 Bleeding7 (7.29)13 (4.76)3 (3.61)0.1776 Perforation4 (4.17)6 (2.20)2 (2.41)0.0728 Fistula1 (1.04)2 (0.73)1 (1.20)0.4492†Wald's Chi-Square test* P < 0.05 statistical significanceData presented as median (range) or number (%).DiscussionThe results of this study confirm Zargar's endoscopic classification of mucosal injuries post caustic ingestion in relation to clinical outcome. Grade 3b mucosal injury assessed by EGD was a predictor of prolonged duration of hospital stay, ICU admittance, and GI and systematic complications. Over 80% of patients with grade 3 burns develop stricture formation, while one-third of those with grade 2 develop pyloric stenosis, acid regurgitation, and perforation [11-13]. In our data, only 50% of patients with grade 3 burns developed stricture formation, while 10% of those with grade 2 developed GI complication. Our lower results may be because of the development and use of more effective anti-acid medications (proton pump inhibitors, H2 antagonists) and more aggressive use of nasogastric irrigation to reduced effect of the substance ingested [6]. The primary reason for ingestion in our patient population was suicidal intent (71%); thus, the injury produced was generally greater and more extensive than that in individuals who ingest caustic substances out of curiosity or by accident [14].Caustic ingestion typically refers to the ingestion of strongly alkaline or acidic household or industrial cleaning products. Alkalis can be found in drain openers, bleaches, toilet bowl cleaners, and detergents containing hydrogen peroxide or sodium hydroxide at concentrations from 4% to 54% [5]. Solid alkaline variants such as crystals or particles adhere to the mucous membrane and increase esophageal injury as a result of prolonged contact with the mucosa [5]. Acid ingestion, which tends to occur less frequently in Western countries (<5%), is more common in countries like Taiwan where hydrochloric acid and sulphuric acid (found in toilet bowl cleaners, antirust compounds, battery fluids, and commercial pesticides) are readily accessible [7].Earlier studies have questioned the recommendation of routine endoscopic evaluation of all patients after presumed caustic ingestion [15,16] on the basis that in the absence of symptoms following unintentional ingestion severe injury is unlikely. The tensile strength of healing tissues in the first 3 weeks is low due to an absence of collagen. New collagen formation does not begin until the second week after injury. Thus, it is advocated that endoscopy should be avoided from 5 to 15 days after caustic ingestion [11]. Currently, EGD evaluation within 12 hours and no later than 24 hours after caustic ingestion is considered safe, and may be beneficial up to 96 hours after ingestion [17,18]. EGD is not recommended from 2 to 3 days up to 2 weeks after caustic ingestion as a result of wound softening.Early classification of caustic substance induced injuries may be beneficial in predicting outcomes [19,20]. Though there are no strict guidelines regarding when endoscopy is indicated, ingestion of larger amounts of corrosives, persistent symptoms, as well as suicidal intention are considered indications for endoscopy in the absence of a third degree burn of the hypopharynx [21,22]. Flexible endoscopy and concurrent endoscopic ultrasound using a high-frequency catheter probe have decreased the rate of perforation that occurs with rigid instruments [8]. This study suggests that patients with mucosal damage exceeding grade 2a are at a higher risk of developing serious complications, while patients with mild mucosal damage have a significantly reduced mortality and morbidity. Death in our patients with grade 2a injury (n = 1) was due to tracheoesophageal fistula, sepsis and acute bleeding; with 2b injuries (n = 2) was lung cancer and sudden apnea, and with 3a injury (n = 1) was hematemesis with sudden apnea. In the patients with grade 2b and 3a injuries, ICU observation and nutritional support may be mandatory if there are any signs of bleeding and the patient experiences abdominal pain, and antibiotics are cautiously recommended in those with lung involvement. Patients with grade 3a lesions may not require immediate surgery [11,23,24].ConclusionIn conclusion, the results of this study indicate that patients with findings of grade 3b burns on endoscopy have high the risk of perforation and complications. Endoscopy done within 12 hours and no later than 24 hours following caustic ingestion to classify mucosal injury subsequent to caustic ingestion is useful to determine the severity of injury, particularly in suicidal cases, and thus helpful in predicting outcomes. A 6-point grading system of mucosal injury, rather than a 4- or 5-point system is useful for predicting immediate and long-term complications, and guiding appropriate therapy.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsC-LC and C-HL carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. J-HT`and Y-YC carried out the immunoassays. P-CC participated in the sequence alignment. N-JL participated in the design of the study and performed the statistical analysis. H-TC conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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batch_10/PMC2533014.json

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+{
+    "id": "PMC2533014",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533014\nAUTHORS: James H Keith, Cheryl A Schaeper, Tresa S Fraser, Malcolm J Fraser\n\nABSTRACT:\nBackgroundThe piggyBac mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of piggyBac, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for piggyBac at amino acid positions D268, D346, and D447.ResultsThis study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the piggyBac transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the piggyBac transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features.ConclusionWe found all the designated DDD aspartates reside in clusters of amino acids that conserved among piggyBac family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.\n\nBODY:\nBackgroundWith the successful application of the Drosophila melanogaster P element as a tool for the creation of transgenic fruit flies, researchers had high hopes for the stable introduction of transgenes into germlines [1]. Unfortunately, P element mobility was determined to be restricted only to D.melanogaster and very closely related species [2]. The ensuing search for novel mobile elements that are active within a wide range of species led to the classification and characterization of the hAT, Tc1-mariner, and piggyBac families of mobile elements [3]. The piggyBac transposon is a short repeat, class II mobile element originally isolated as the causative agent of the Few Polyhedra plaque morphology mutations of Autographa california nuclear polyhedrosis virus (AcNPV) [4]. It is the archetype of its own family and the only fully active transposable element in that family that is currently in use as a transgenic vector. The wild-type piggyBac element is 2.4 kb long and ends in a 5' CCC...GGG 3' configuration with asymmetric terminal and subterminal repeats. The sub-terminal repeats are 19 bp in length and lie 3 bp and 31 bp inside of the perfect inverted terminal repeats (ITRs) on the 5' and 3' end respectively [4]. The protein catalyzing the movement of piggyBac is encoded within a single open reading frame (ORF) of 1783 bp, with a length of 594 amino acids and a predicated mass of 68 kDa [4,5]. As a class II mobile element, piggyBac operates via a DNA intermediate in a cut-and-paste mechanism [6]. In this scenario, the transposable element, delineated by transposase-specific ITRs is excised by the transposase and reinserted into a new location. In the case of piggyBac, the bias for individual sites chosen during reinsertion does not follow any clearly defined rules. Although the only sequence requirement for a new insertion event is the presence of a TTAA sequence in the DNA, there is a clear preference for certain TTAA sites over others as shown by inter-plasmid transposition assays [7].The piggyBac element has been used to transform the species Mus musculus [8], Tribolium castaneum [9], Anopheles gambiae [10], Ceratitis capita [11], Drosophila melanogaster [12], Bactrocera dorsalis [13], Musca domestica [14], Lucilia cuprina [15], Bicyclus anynana [16], Aedes aegypti [17,18], Anopheles albimanus [19], Anopheles stephensi [20], Bombyx mori [21], Athalia rosae [22], Drosophila willistoni [23], Pectinophora gossypiella [24], Anastrepha suspensa [25], Aedes fluviatilis [26], Harmonia axyridis [27], Schistosoma mansoni [28], and the causative agent of malaria, Plasmodium falciparum [29]. Plasmid-based mobility assays have also shown piggyBac to be active in human and other primate cells [8,30], in Zea maize cells [31], Spodoptera frugiperda cells [5], and in the embryos of Aedes triseriatus [32], Heliothis virescens [33], and Danio rerio [30], although none of these species have yet been transformed with piggyBac.Recently, Mitra et al. [34] have defined piggyBac mediated transposition as involving several discrete steps. First, the transposase creates 3' nicks at each end of the terminal repeats just internal of the 'TTAA'. The free 3'-OH attacks the complimentary strand, four nucleotides downstream of the initial nick, just outside of the 'TTAA' repeat, forming a transient hairpin structure and releasing the transposon ends. The transposase quickly resolves these hairpin structures into 5' 'TTAA' overhangs on either side of the excision product. For reinsertion, the transposase joins the excised element to a new 'TTAA' target site in a staggered pattern, completing the transposition reaction. This mechanism accounts for both precise excision and target site duplication.For all transposable elements, there are certain requirements for transposition, namely the recognition and binding of the terminal repeats, double stranded DNA breakage (DSB), and reinsertion of the element into a target, as well as possibly the repair of the excision site, which may or may not involve host factors [3]. DNA manipulating enzymes, such as integrases, recombinases, resolvases, endonucleases, and transposases must meet one or more of these requirements in order to successfully catalyze their respective reactions. With this commonality in function comes some similarity in mechanism and structure. One of the most widespread of these features is the 'DDE/DDD' motif first identified and designated in retroviral and retrotransposon integrases [35]. This motif has since been identified among many different families of DNA manipulating enzymes, from certain nucleases to the enzymes that carry out V(D)J recombination and even the human RNAi enzyme, Ago2 [36-47]. 'DDE/DDD' has been implicated as the active site for such enzymes by providing carboxyl groups used to recruit metal ions as essential cofactors. These residues are required to precisely position the metal ions, usually magnesium or manganese, used to recruit nucleophilic groups to the catalytic center of their respective enzymes.Sarkar et al. published an extensive computational analysis of the piggyBac element and related sequences [48] that demonstrated the widespread occurrence of the piggyBac family of elements. While no actual experiments were performed by Sarkar et al., they proposed a 'DDD' motif for piggyBac, similar to a paper published in 1996 in which Bigot et al. speculated the existence of a 'DSE' motif in hAT elements based on sequence alignments. Sarkar et al. based their proposal on weak matches for two piggyBac related proteins to the bacterial IS4/5 transposase family DDE motif. This alignment suggested the first two of these aspartates are D268, and D346. While this similarity with the IS4/5 transposases did not yield a hit for the third member of the 'DDE' triad, Sarkar et al. speculated that the third member was an aspartate at D447.In this report, we align very closely related members of the piggyBac family across kingdoms and identify several highly conserved regions. We propose that a direct relationship exists between the degree of conservation of a particular feature, and its necessity to catalyze transposition, as they are favored at the time of horizontal transmission. To this end, we only considered the proteins most closely related to piggyBac in our alignment. However, it should be made clear, when considering the results of our alignment, aside from piggyBac itself, only one other family member has been shown to be a functional element: Xtr-Uribo2_PCR_Iv1b (Genbank Accession: BAF82025) from the Xenopus family, which catalyzes movement in GP293 cells [49]. Therefore, we are careful to judge the relevance of any well conserved feature primarily upon its presence in either piggyBac or Uribo-2.Finally, we mutate the three aspartates identified by Sarkar et al. as well as D450, another highly conserved residue close to the third proposed aspartate, which may also contribute to the catalytic center of the transposase. We test these site-directed mutant transposases for excision activity in two different donor plasmids and report our results.ResultsSequence alignmentWe performed a GenBank [50] search for proteins closest in similarity to the wild-type Trichoplusia ni piggyBac transposase sequence. We chose hits from a wide variety of organisms for a sequence alignment by ClustalW (Table 1). We then exported the sequence alignment file and entered it into the BoxShade server [51] to more easily visualize any conserved residues or groups of residues.Table 1Proteins used in the alignmentAccessionTaxonProtein NameJ04364Trichoplusia nipiggyBacAAM76341Daphnia pulicaraPokey 6.6 kbAAM76342Daphnia pulicaraPokey 5 kbABS18391Helicoverpa armigeratransposaseBAD07480Bombyx moriYabusame-wBAD11135Bombyx moriYabusame-1BAD11136Bombyx moriYabusame-2BAF82017Xenopus laevisKobutaBAF82019Xenopus tropicalisUribo-1BAF82020Xenopus laevisUribo-1BAF82021Xenopus borealisUribo-1BAF82022Xenopus tropicalisUribo-2CAF99963Tetraodon nigroviridisUnnamed ProductCAI18712Homo sapiensPGBD-2NP_689808Homo sapiensPGBD-4NP_741958Mus musculusPGBD-5Q8N328Homo sapiensPGBD-3Q8N414Homo sapiensPGBD-5Q96JS3Homo sapiensPGBD-1XP_001030237Tetrahymena thermophilaPredicted proteinXP_001541487Ajellomyces capsulatusPredicted proteinXP_001599370Nasonia vitripennisPredicted proteinXP_312615Anopheles GambiaePGBDXP_590785Bos taurusPredicted proteinXP_699416Danio reroPredicted proteinXP_797885Strongylocentrotus purpuratusPredicted proteinGenBank accession number, taxon, common name, and protein name of the proteins used in the ClustalW alignment.The sequence alignments (Fig. 1, 2, 3, 4, 5, 6, 7) immediately revealed several highly conserved features. Starting at the N-terminus of the transposase alignment, we noticed four conserved acidic residues: D32, D38, E45, and D49 (Fig. 1). The presence of either an aspartate or a glutamate at these positions does not seem to matter even within families; the acidic charge is the only conserved feature we can distinguish.Figure 1Alignment of piggyBac-related proteins. A BoxShade server alignment of the proteins listed in table 1. Residues aligning with piggyBac position 1–63 are shown. Only two piggyBac family proteins have been shown to catalyze transposition, these are indicated by bold face type and asterisks.Figure 2Alignment of piggyBac-related proteins. A BoxShade server alignment of the proteins listed in table 1. Residues aligning with piggyBac position 64–160 are shown. Only two piggyBac family proteins have been shown to catalyze transposition, these are indicated by bold face type and asterisks.Figure 3Alignment of piggyBac-related proteins. A BoxShade server alignment of the proteins listed in table 1. Residues aligning with piggyBac position 161–260 are shown. Only two piggyBac family proteins have been shown to catalyze transposition, these are indicated by bold face type and asterisks.Figure 4Alignment of piggyBac-related proteins. A BoxShade server alignment of the proteins listed in table 1. Residues aligning with piggyBac position 261–351 are shown. Only two piggyBac family proteins have been shown to catalyze transposition, these are indicated by bold face type and asterisks. The aspartate residues mutated in this study are highlighted in yellow.Figure 5Alignment of piggyBac-related proteins. A BoxShade server alignment of the proteins listed in table 1. Residues aligning with piggyBac position 352–451 are shown. Only two piggyBac family proteins have been shown to catalyze transposition, these are indicated by bold face type and asterisks. The aspartate residues mutated in this study are highlighted in yellow.Figure 6Alignment of piggyBac-related proteins. A BoxShade server alignment of the proteins listed in table 1. Residues aligning with piggyBac position 452–558 are shown. Only two piggyBac family proteins have been shown to catalyze transposition, these are indicated by bold face type and asterisks.Figure 7Alignment of piggyBac-related proteins. A BoxShade server alignment of the proteins listed in table 1. Residues aligning with piggyBac position 559 through the c-terminus are shown. Only two piggyBac family proteins have been shown to catalyze transposition, these are indicated by bold face type and asterisks.The core of the transposase possesses clusters of conserved amino acids with very well defined borders for each conserved stretch. The first conserved cluster we identified begins shortly downstream of a conserved proline, P131, and may mark the beginning of a functional domain in piggyBac transposase family members. The next clusters of conservation lie roughly from P131 – N152, E175 – K190, and V207 – D228 (Fig. 2, 3). The region between D239 – Y259, while not particularly conserved in piggyBac itself, is conserved in other members of the family. Additionally, the next proline from this cluster, P261, starts another highly conserved region which includes the first member of the proposed 'DDD' triad, D268 (Fig. 4).The next group of conserved residues contains the second aspartate, D346 and an absolutely conserved glycine at position 369. G369 marks the start of the well conserved motif 'GTVRxNKRxIP' (Fig. 5). This motif ends with P379, possibly delineating the end of a functional domain. This motif is not present in its entirety in Uribo-2. However, the analogous sequence of Uribo-2 'GTINRNRKxLP' does preserve similar features.Downstream from this region is another conserved group that starts with a highly conserved proline at position 433. P433 is only a short distance from D447 and D450, the last two aspartates tested in our study, and is the start of another motif: 'PxxxxxxYxxxxGGVDxxD.' The aspartates in this motif are D447 and D450, respectively (Fig. 5, 6).A cysteine-rich domain overlapping with the piggyBac nuclear localization signal (NLS) is positioned at the C-terminus of piggyBac. The spacing between these cysteines is well conserved among piggyBac family members when present, even though the intervening amino acids are not. Additional cysteines also lie within this region, conforming to a novel zinc-finger domain in piggyBac [52,53]. A basic amino charge at R568 is also well conserved (Fig. 7).Excision assayThe excision activity of a nonautonomous piggyBac element from the donor plasmids pBKOα [32] was assessed for 8 mutant transposases relative to the wild-type transposase (Fig. 8; Fig. 9). Plasmid DNA was extracted from transfected cell cultures and transformed into E. coli cells. As with previous assays [32], the number of non-excised plasmids was reduced by restriction digestion of a site unique to the donor element. The large number of colonies precluded analysis of every transformant, but a representative cross section was chosen for analysis by restriction enzyme digestion. Positive events were further screened by sequence analysis. Only events that showed precise excision were tallied.Figure 8Diagram of piggyBac ORF and genotypes used in this study. A diagrammatic representation of the piggyBac transposase mutant genotypes used in this study. The entire piggyBac ORF is displayed with the aspartates noted with their corresponding residue number. 4 × NLS represents 4 PSORT predicted nuclear localization patterns as described in discussion.Figure 9Schematic of the REN colony screen. (A) The donor plasmid pBKOα is co-transfected with one of the piggyBac expression plasmids (fig. 8), transformed into E. coli and plated. (B) Precise excision of the piggyBac cassette removes the lacZ open reading frame. Digestion with BglII removes any excess piggyBac expression plasmids and donor plasmids which have not undergone excision. Excision events are scored as white colonies. Plating a portion of undigested plasmids and scoring blue colonies yields the total number of potential donor plasmids.In order to obtain the largest amount of data and to ascertain if differences in the donor plasmid had any effect on excision efficiency, a second assay employing a blue/white colony screen was developed using the Topo-TA vector backbone, pCR2.1-Topo (Invitrogen, Carlsbad, CA) to make the donor plasmid pCR2.1-piggyBac{SV40}. Precise excision events would restore the 'TTAA' sequence of the region between the two lacZ coding sequence fragments, allowing for the expression of a fully active β-Gal protein. This assay allowed rapid screening of large numbers of colonies and identified positive excision events (Fig. 10). A simple PCR of blue colonies confirmed that excision had occurred (Fig. 11). Subsequent sequencing of some of these blue colonies validated these findings.Figure 10Schematic of the blue/white colony excision assay. (A) The donor plasmid pCR2.1-piggyBac{SV40} is co-transfected with one of the piggyBac expression plasmids (fig. 8) transformed into E. coli and plated. (B) Precise excision of the piggyBac{SV40} cassette restores the original lacZ open reading frame at the original 'TTAA' insertion site. By plating on kanamycin media containing IPTG and X-gal, only E. coli containing donor plasmids will develop into colonies. Colonies which are blue represent excision events, while the number of blue and white colonies represents the total number of potential donor plasmids.Figure 11PCR analysis of donor plasmid sizes from the blue/white colony screen. PCR of 10 white (negative, top row) colonies and 10 blue (positive, bottom row) colonies, randomly chosen, from the blue/white colony screen is shown on a .9% TAE agarose gel stained with ethidium bromide. The amplicon crosses the entire donor fragment. Expected band size for the pre-excision plasmid is 1027 bp and 202 bp for the post-excision donor plasmid amplicon.Our mutational analysis demonstrates that all members of the proposed triad, D268, D346, and D447, are required for efficient piggyBac catalyzed excision in a cellular environment. The additional amino acid targeted for mutational analysis, D450, has a primary requirement for a negative charge, since the asparagine replacement significantly inhibited excision, while a glutamate replacement yielded moderate excision, although not to the degree of the wild-type transposase.Even though the trends remained the same, a difference in overall excision efficiency between donor plasmids was also demonstrated. The pBKOα plasmid donor element was able to facilitate some level of excision for all mutant transposases except D447N (Fig. 12). In contrast, despite the larger number of colonies screened, pCR2.1-piggyBac{SV40} excision failed to yield events for D268E, D268N, D346E, and D447N (Fig. 13). When precise excision was catalyzed, all frequencies were less than that of the pBKOα counterpart when the same helper plasmid was utilized (Table 2). Incomplete digestion of the helper plasmid in the REN screen excision assay prior to plating (see: methods) may have been a contributing factor, leading to artifact colonies which would inflate the actual number of excised colonies. However, excision efficiency may be influenced by other factors, such as donor fragment size or the physical properties of the DNA at the excision sites. The size of the excision fragments of the two donor plasmids vary considerably: 6218 bp for pBKOα and 825 bp for pCR2.1-piggyBac{SV40}.Figure 12Relative frequency of excision obtained with the REN colony screen. Positive control, wild-type piggyBac, is set to 100. Data are expressed as a mean of three replicates +/- standard error bars. ANOVA test was performed using GraphPad Prism 3.0 software. Means were considered statistically significant when p-values less than 0.05 were obtained with the Dunnett's post-test. Statistical significance of difference with regards to positive control is indicated on all data figures as asterisks above bars. (p < 0.05)Figure 13Relative frequency of excision obtained with the Blue/White colony screen. Positive control, wild-type piggyBac, is set to 100. Data are expressed as a mean of three replicates +/- standard error bars. ANOVA test was performed using GraphPad Prism 3.0 software. Means were considered statistically significant when p-values less than 0.05 were obtained with the Dunnett's post-test. Statistical significance of difference with regards to positive control is indicated on all data figures as asterisks above bars. (p < 0.05)Table 2Excision frequencies for donor plasmidsBlue-WhiteREN ScreenMutantExcision Frequency+/- SEExcision Frequency+/- SED268E0.00E+000.00E+009.96E-033.73E-03D268N0.00E+000.00E+008.03E-054.73E-05D346E0.00E+000.00E+008.97E-054.90E-05D346N2.97E-051.49E-053.62E-042.02E-04D447E4.44E-058.54E-062.40E-041.84E-04D447N0.00E+000.00E+003.99E-042.93E-04D450E8.80E-041.36E-040.00E+000.00E+00D450N1.38E-051.38E-051.25E-030.00E+00+ctrl1.40E-022.12E-033.15E-038.86E-04-ctrl0.00E+000.00E+0000E+000.00E+00Values obtained for the frequency of excision using the two different donor plasmids with each of the mutant transposases. These numbers are normalized to a positive control frequency of 100%.Western blotA western blot was performed to ensure that full length piggyBac transposase was being produced from the expression vectors. This was done to verify that any mutant expression vectors unable to drive excision activity were a result of the mutations in the transposase and not for a lack of expression through faulty vectors. All mutant vectors expressed equivalent amounts of protein of approximately 68 kDa to which a polyclonal piggyBac antibody was able to bind. All lanes probed positive for actin, used as a loading control (Fig. 14).Figure 14Western blot of piggyBac mutant transposases. A western blot was performed on each piggyBac mutant with 125 μg total cell lysate per lane. The top row was probed with anti-piggyBac antibody and indicates the presence of the piggyBac transposase at 68 kDa in each mutant, the positive control, phsp-pBac, and a lack of transposase in the negative control. The bottom row was probed for actin, a 43 kDa protein, using anti-actin I-19 and shows equal loading in all lanes.DiscussionSarkar et al. suggested the presence of a 'DDD' motif for piggyBac, without empirical evidence of the association of the motif with mobility. Their proposal was based on two weak matches returned when two piggyBac related proteins, but not piggyBac itself, were used as a query in an NCBI Conserved Domain Search. These query hits were for the Pfam domain pfam01609 [48]. The conserved D268 and D346 were thus matched to the bacterial IS4/5 transposase family DDE motif based on their degree of conservation in the transposase core and the presence of a glutamate immediately following D268, and an asparagine following D346. No reason for choosing D447 was cited, but both D447 and D450 are part of a highly conserved motif discussed below.Bigot and colleagues [54] used a similar approach of transposase alignments to propose the existence of a 'DDE' motif in hAT elements, with the second aspartate replaced by a serine in Ac, hobo, and Hermes. They aligned members of the Tc1-mariner superfamily with members of the hAT family and identified conserved and similar residues common between both groups. The 'DSE' speculation has since been empirically disproven [55]. However, of this 'DSE' triad, both D402 and E572 (with respect to Hermes) were essential for transposition while alteration of S535 to either an alanine or an aspartate had no statistically significant effect on transposition efficiency. This, at least, demonstrated the necessity for D402 and E572, but did not prove if either residue was part of an essential triad.Starting at the N-terminus of piggyBac there are four acidic amino acids: D32, D38, E45, and D49 that are present in most of the aligned proteins (Fig. 1). While charges are conserved at these positions, the residues themselves do not seem to have any requirement as to whether they are an aspartate or a glutamate. Interestingly, this interchangeability is particularly variable at these positions, even within closely related proteins, such as the piggyBac related proteins in three different species of Xenopus, and among the human piggyBac derived proteins (PGBD). Examination of piggyBac related transposons in Xenopus identified three elements, Uribo-1, Uribo-2, and Kobuta. Xtr-Uribo2_PCR_Iv1b proved to be a functional mobile element complete with transposase able to catalyze movement in GP293 cells [49]. Kobuta, however, lacks excision activity. Xtr-Uribo2_PCR_Iv1b possesses a glutamate at piggyBac's D32 and does not have a match for E45, even though other inactive Xenopus putative transposases, Uribo-1 and Kobuta, do have matches for E45. Additionally, these N-terminal acidic residues are spaced so closely together that they are unlikely to be the DDD/DDE triad in our opinion. Interestingly, both Uribo proteins contained analogs to D268, D346, and D447, while the inactive Kobuta protein contained only D346 as a rule with two divergent Kobuta examples also having the D268 residue. All Xenopus proteins also harbor the highly conserved D450 residue.Distinct clusters of conserved amino acids are present through the rest of the piggyBac family starting at P131 (Fig. 2). The fact that the first constellation of conservation begins with a proline is worth noting, as proline is known to disrupt the periodic structure of α-helices and β-sheets, often demarcating the protein from one functional domain to the next [56]. Together with the adjacent region of conservation, we speculate that the area just downstream of P131 is most likely a functional domain in the transposase. A conserved domain search returns an extremely weak (e = .44) match to pfam02388, Staphylococcus proteins involved in formation of the peptidoglycan layer, a coincidence in our opinion.The piggyBac family analog to K246 is also a highly conserved proline, but as this amino acid is not present in piggyBac it is not required for a functional transposase (Fig. 3). However, P261, while less conserved than the K246 proline analogs, is present in both piggyBac and Uribo-2. It also lays just N-terminal of the very well conserved residues D268, the first member of the 'DDD' triad, and E269.The next cluster of conserved amino acids includes D346, the second member of the proposed 'DDD' triad (Fig. 4). Also in this region lies the only residue in the alignment that is absolutely conserved is G369 (Fig. 5). This glycine is the start of a nearly universally conserved motif among piggyBac related proteins: 'GTVRxNKRxIP.' While R372 is limited to arginine, the other two basic amino acids, K375 and R376, seem to be conserved only in charge, as some proteins use either arginine or lysine at these positions. When a basic amino acid occupies sites analogous to both position 375 and 376 in a protein, it is always one of each and never the same residue, except for Strongylocentrotus purpuratus which utilizes arginines in both locations. I378 is also another residue conserved only in properties, in this case hydrophobicity. Methionine, leucine, and isoleucine each are employed at this site with no clear pattern as to which is used outside of immediately related members. Finally, P379 does not seem to indicate the start of any highly conserved clusters, but it does lie at the end of a conserved cluster starting at approximately E331, possibly ending a functional domain in piggyBac. Of note, this motif is not present in its entirety in the functional transposase, Uribo-2. However, the analogous sequence of Uribo-2 'GTINRNRKxLP' does preserve similar features.The next highly conserved proline, P433, is located only a short distance from P379, just upstream of the third proposed aspartate, D447, and another highly conserved aspartate at D450 (Fig. 5). This proline delineates the start of one of the most highly conserved motifs we found in our alignment: 'PxxxxxxYxxxxGGVDxxD' which contains both D447 and D450. This motif is present in both piggyBac and Uribo-2 as well as most other members of our analysis. Among members where this motif is loosely present, G444 is replaced by an alanine in the silkworm Yabusame proteins, and G445 is replaced with an alanine in the human PGBD-4. P433 could mark the start of another required functional domain in piggyBac, and for this reason we decided to mutate both D447 and D450. In the more divergent proteins, mouse PGBD-5, human PGBD-5, human PGBD-1, Anopheles gambiae PGDB protein XP_312615, and Ajellomyces capsulatus predicted protein XP_001599370, this motif is not fully present.The C-terminus of most piggyBac related proteins contains a cysteine-rich motif: C559, C562, C574, C577, and C582 (Fig. 7). The spacing between these residues is somewhat conserved even though the intervening residues are not. In piggyBac, this region is also a novel match for a RING-finger motif [52], a type of Zinc-finger which has been implicated in protein-protein interactions [53]. The high degree of conservation in this region suggests a selective pressure for the presence and spacing of these cysteines. Recently, Mitra and colleagues [34] have suggested this Zinc-finger could in fact be a PHD finger, implicated in heterochromatin interactions. The ability of their purified transposase to function in an in vitro environment on free, unwound DNA despite the removal of this C-terminal domain is consistent with this hypothesis. The occurrence of a basic charged residue at R569 is also a commonality.In piggyBac, we have demonstrated that this area contains a functional NLS (aa 551–571) (in press). In fact, this region is so rich with basic amino acids, that 4 PSORTII [57] predicted NLS matches exist in this short region: 2 monopartite at positions 551 and 563, and 2 bipartite signals at positions 554 and 555, respectively. Interestingly, this NLS cluster is not present in the functional Uribo-2 transposase, however a PSORT analysis of Uribo-2 shows it harbors a NLS matching a monopartite pattern beginning at P276, with respect to the Uribo-2 sequence.The excision assays reveal that substitution of D268, D346, D447, or D450 with either an asparagine or a glutamate impairs the function of the transposase from the donor plasmid pCR2.1-piggyBac{SV40}. In contrast, while the substitution D450E significantly impaired excision from pCR2.1-piggyBac{SV40}, it did not appear to have a statistically significant effect with regards to pBKOα. The reasons for this apparent difference reflect level of error inherent in the protocols between the two assays. For the REN colony screen, a randomly chosen representative cross section is cultured and digested from the colonies available. This method is prone to a larger amount of error as fewer colonies are screened for excision events. However, the blue/white colony screen permits the analysis of all the colonies on a plate for excision events. This provides a much larger population of donor plasmids to be analyzed and reduces the standard error.These four residues mutated in our study are far from the only conserved acidic amino acids in the piggyBac family alignment: the short cluster at the amino terminus previously discussed, as well as D141, E175, D249, and D300 are all conserved to a degree, and may be involved in transposition. Each of the mutations tested severely reduced or completely shut down excision activity of the transposase. Previous studies of acidic amino acids in Hermes support the idea that essential amino acids are not limited to just the three members of a proposed triad, as alterations to D180, D248, D402, and E572 in the Hermes transposase all affect transpositional activity [55,58].Here, we test only the excision step of the entire transposition reaction in a cell culture system. There are other possibilities that cannot be ruled out by our data, including the necessity of these residues in forming a functional secondary structure of the transposase. We cannot say for certain which specific parts of the excision process these mutations hinder. It is entirely possible that they are required for DNA binding or possibly the recruitment of auxiliary host factors. However, the reports of Mitra et al. [34] examining piggyBac in function an in vitro system suggests that the transposase alone may carry out all steps of transposition without any additional requirement for host factors. Substitution of D268, D346, and D447 with alanine does not inhibit specific binding of the transposase to the terminal repeats of piggyBac [34], but each of these mutations abrogates all steps of transposition, including 3' OH nicking and hairpin formation – both integral steps for excision. Furthermore, unlike wild type piggyBac, these mutants are defective for target joining when synapsed with an artificial excision fragment. Testing these mutants in a yeast system also demonstrated they were functionally defective [34]. The lack of catalytic activity in our D268, D346, and D447 mutant assays confirms the relevance of the observation of Mitra et al. to piggyBac movement in insect cell systems.In contrast, Mitra et al. demonstrate that D450A is still catalytically active in an in vitro environment, while our results show a definite interfering effect. One explanation for this discrepancy is that D450 is likely not a part of the metal interaction motif, but may be involved in the proper folding of the protein with limited tolerances for certain amino acids. Certainly, our findings support that D450 is the least critical of the four aspartates we tested, but is still necessary to a degree.Just because an amino acid is acidic and required for transposition does not automatically make it a member of a divalent metal interaction motif, nor does particular mutant interfere with all steps of transposition. An example of this can be found in a mutational study of the V(D)J recombination initiator, RAG1. In this experiment, a great number of acidic amino acids were mutated, some of which impaired cleavage activity. One of these mutations, E811Q, was deficient for DNA binding activity, a loss-of-function that could indirectly lead to decreased cleavage activity. This study also defined two classes of mutants: class I which retained a measure of cleavage activity, and class II which yielded no detectable cleavage products, even though they retained DNA binding activity [42]. It is possible that alteration of D450 in piggyBac may be analogous to such a class I mutant.Another example of a mutant transposase deficient for specific steps of transposition would be the D248A mutant of Hermes, which was deficient for DSB activity. Upon further investigation, this mutant appeared to only be deficient in the first 5' nicking step of excision. When supplied with a pre-nicked substrate, it was able to facilitate hairpin formation and a measure of target joining in the presence of Mn2+, the second step of DSB and the final step of transposition, respectively [58]. These examples illustrate that a number of steps exist in which a mutant could interfere with transposition, thereby decreasing the formation of the end product.Iron-induced hydroxyl radical protein cleavage is one direct test for metal binding activity [59]. This assay takes advantage of the ability of Fe2+, in the presence of ascorbate or H2O2, to generate hydroxyl radicals. If the protein in question has the ability to bind Fe2+, then hydroxyl radicals will be generated at the sites where the ion is located. These radicals subsequently cleave the peptide backbone of the protein which bound the Fe2+, yielding cleavage products with sizes consistent with the location of the metal binding pockets. Regarding recombinases, this approach was used to narrow down the location of the metal binding residues of RAG1 prior to the generation of site-specific mutants [43].Another common test for metal interaction is the replacement of either the aspartate or the glutamate with a cysteine [60]. This test is based on the ability of glutamate and aspartate metal ligand residues to supply a carboxyl group for metal interaction. These amino acids use the oxygen on their R-chain as the interacting atom via O-Mg2+ or O-Mn2+ formation. However, interactions with Mn2+, but not Mg2+, are also able to occur using a cysteine via a S-Mn2+ bond [61]. Such an interaction can be analogous to the metal binding activities of glutamate and aspartate. For example, substitutions D180C, D248A, and D248C in Hermes all showed the ability to catalyze hairpin formation of substrate DNA in the presence of Mn2+ during the excision step of transposition, but only D180C and D248A were able to complete the joining of the transpososome to target DNA. E572A and E572C left Hermes unable to facilitate any step of the transposition reaction [58].Finally, much work has been done with X-ray crystallography on integrases, resolvases, and transposases at different stages of catalysis, including multimers, free floating proteins, and enzymes complexed with their target DNA. Direct resolution of these structures has been invaluable to the understanding of structure-function relationships. Indeed, the various studies have found structural similarities in the active sites of the recombinase family and nucleases, including the DDD/DDE metal binding motif for both one and two metal binding proteins [62-69]. In light of what we and others have found [34] it would not be surprising to find piggyBac shared many features common to the recombinase family.ConclusionThe piggyBac family alignment revealed a number of interesting features in the transposase. Regions of high conservation in the catalytic core were sometimes demarcated by proline residues, possibility separating functional domains. Furthermore, we found four conserved acidic residues at the N-terminus of the transposase, and four more throughout the rest of the transposase. A cysteine-rich region with somewhat conserved spacing exists at the C-terminus and is a novel match for the RING finger protein-interaction motif. Our data indicate which of the four tested residues are essential for transposase-mediated excision from a donor plasmid in Drosophila cultured cells. Our donor plasmids yielded similar results, however a charge-preserving substitution from aspartate to glutamate was at least moderately tolerated at positions D450. The existence of an acidic charge was necessary at all four positions tested for both of our donor plasmids with a specific requirement for an aspartate at D268, D346, and D447. We conclude that the four amino acids tested are indeed vital for efficient excision of the piggyBac element from a donor plasmid in cell culture.MethodsSequence retrieval and alignmentThe predicted piggyBac translated protein was used to scan GenBank [50] in a protein-BLAST search of the non-redundant database. Hits that were not from Trichoplusia ni were chosen for a multiple sequence alignment in ClustalW [70]. The resulting .msf alignment was input into the BoxShade server in order to more easily identify the conserved regions.Plasmid ConstructionpXL-BacII-SV40 was constructed by amplifying the SV40 terminator sequence from pMT/V5-HisA (Invitrogen, Carlsbad, CA) with the primers: Sense: 5' TGTGCGGCCGCAGTCTAGAAAAGGATCCTAGATCATAATCAGCCATACCACATTTGTAGAGG 3' and antisense: 5' TGGGAGCTCATAAGCCGTATCGATAAGCTTTAAGATACATTGATG 3' with Pfx high-fidelity polymerase (Invitrogen) according to manufacturer protocol. The PCR reaction was separated on .9% agarose gel stained with 10 μg/ml ethidium bromide and the band corresponding to the SV40 fragment was isolated with the Wizard SV Gel Purification System (Invitrogen). The fragment was then cut with BamHI (New England Biolabs (NEB), Ipswitch, MA) and ClaI (NEB) and ligated into the vector pXL-BacII [71] at the same sites to form pXL-BacII-SV40. The plasmid was sequenced to verify accuracy.To create pCR2.1-piggyBac{SV40}, the piggyBac{SV40} fragment was amplified from pXL-BacII-SV40 with the primers: Sense: 5' AAACCCAAAGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGGGGTTAACCCTAGAAAGATAATCATATTGTGACG 3' and Antisense: 5' CCCAAACCCGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGGGGTTAACCCTAGAAAGATAGTCTGCGTAAAATTG 3' using Pfx high-fidelity polymerase. The 'TTAA' sites delineating the ends of the piggyBac donor fragment are in bold while the sequences remaining behind after precise excision are underlined. The fragment corresponding to piggyBac{SV40} was band-isolated as described above and cut with the restriction enzymes Acc65I (NEB) and NotI (NEB) This fragment was then ligated into the vector pCR2.1-Topo (Invitrogen) at the same sites. The plasmid was sequenced to verify accuracy.The various mutants of the piggyBac open reading frame were prepared by Bio Basic Inc. (Biobasic Inc., Ontario, Canada) in the vector backbone phsp-pBac [12] according to the specifications listed in Table 1.Blue/White colony screenThe blue/white colony screen was developed to enable us to determine, by visual inspection, which colonies had undergone precise excision. The piggyBac{SV40} cassette was inserted at a 'TTAA' site in the pCR2.1 backbone, in the midst of the lacZ ORF. The piggyBac expression plasmid and the pCR2.1-piggyBac{SV40} are co-transfected into D. melanogaster Schneider2 (S2) cells and incubated. The plasmids are recovered by Hirt extraction and transformed into E. coli. In the presence of the cassette, the lacZ ORF is interrupted, yielding a white colony phenotype on kanamycin containing media with IPTG and 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal). However, when precise piggyBac-catalyzed excision occurs in this vector, the lacZ ORF is restored and the subsequent E. coli colony will possess a blue phenotype (Fig. 10). Plating on kanamycin containing media ensures that only donor plasmids will develop into colonies, and helper expression plasmids will not lead to false white colonies. By comparing the number of blue colonies to the number of total (blue+white) colonies, we can extrapolate the excision frequency. That is, the number of excision events occurring out of the total number of potential excision events.106 S2 cells were allowed to adhere to the bottom of a 9.6 cm2 cell culture well and were transfected with .4 μg of phsp-pBac helper plasmid and .4 μg of pCR2.1-piggyBac{SV40} donor plasmid with Transfectin liposomal transfection reagent (Bio-Rad Laboratories, Hercules, CA) according to manufacturer protocol in minimal S2 media (Gibco, Carlsbad, CA). The cell/transfection media mixture was allowed to sit at room temperature overnight. The next morning, the transfection media was aspirated from the cells and replaced with complete S2 media (Minimal S2 supplemented with 10% FBS, .1 mg/mL streptomycin, and .25 μg/mL amphotericin). The cells were heat-shocked to induce phsp-pBac promoter activation at 37°C for 60 minutes. The cells were then moved to a 28°C incubator for 48 hours prior to harvesting.Plasmid DNA was isolated from the S2 cells according to the standard Hirt LMW DNA extraction protocol [72]. Additional steps taken were as follows: Following the centrifugation of the lysate, the DNA-containing supernatant was removed, extracted once in phenol:chloroform:isoamyl alcohol (25:24:1) and washed once with chloroform:isoamyl alcohol (24:1). DNA was precipitated with 2 volumes 100% ethanol and 1/10 volume 3 M sodium acetate (pH 4.5) and stored at -20°C overnight. DNA was pelleted by centrifugation at 14,000 rpm at 4°C and the supernatant aspirated. The pellet was washed 3 times with 70% ethanol to remove the excess salt from the Hirt extraction buffers, dried, and resuspended in 40 μl of nuclease-free water. 1 μl of the Hirt extraction DNA was mixed in 21 μl nuclease-free water and transformed by electroporation into 3 μl E coli. DH10B cells (Invitrogen) in a 1 mm gap electroporation cuvette according to manufacturer protocol. 150 μl of S.O.C. medium was pipetted directly into the electroporation cuvette and mixed well by pipetting. Cells were allowed to recover at 37°C for 30 minutes prior to plating. 10 μl and 100 μl portions of this cell-media mixture were spread onto LB-Kanamycin plates prepared with 4 μl 100 μg/μl IPTG and 70 μl 20 mg/ml X-gal. The plates were incubated overnight at 37°C and scored by counting the number of blue colonies on the plate spread with 100 μl cell-media mixture and the total number of colonies on the plate spread with 10 μl of the cell-media mixture.PCR verification of excision eventsA portion of the blue colonies were screened for excision events to verify accuracy of the test. Briefly, a 20 μl total volume PCR was prepared with a final concentration of 50 mM KCl, 10 mM Tris pH 8.3, 1.5 mM MgCl2, 5 units of Taq polymerase, and 10 pmol of each standard M13 primers: sense 5' GTAAAACGACGGCCAGTG 3' and antisense: 5' GGAAACAGCTATGACCATG 3'. The colony was taken directly from the plate and resuspended into the 20 μl PCR reaction by pipetting. 10 μl of the thermocycled PCR reaction was separated on .9% agarose stained with 10 μg/ml ethidium bromide.Restriction analysis colony screenThe REN digestion excision assay follows pre-established methods, using the plasmid pBKOα [32] (Fig. 9). pBKOα and a piggyBac expression helper plasmid are cotransfected into S2 cells and incubated. Plasmid DNA is recovered by modified Hirt extraction as detailed in the blue/white colony screen section of methods and transformed into E. coli cells. A portion of this recovered plasmid is digested with BglII to remove any helper and non-excised donor plasmids and plated on LB-Amp/X-gal/IPTG plates. Because excision removes the lacZ gene, white colonies are scored as excision events. A portion of the undigested Hirt extraction is also transformed and plated on LB-Amp/X-gal/IPTG plates and the number of blue colonies is scored as the total number of potential donor plasmids.106 S2 cells were allowed to adhere to the bottom of a 9.6 cm2 cell culture well and transfected with 0.5 μg of phsp-pBac helper plasmid and 0.5 μg of pBKOα donor plasmid with Transfectin liposomal transfection reagent (Bio-Rad) according to manufacturer protocol in minimal S2 media (Gibco). Two wells were transfected per helper-donor pair. The transfection media remained on the cells overnight and was replaced with Schneider's complete media. At 24 hours post transfection the cells were heat shocked for 1 hour at 37°C. At 24 hours post heat shock the cells were harvested by Hirt extraction as detailed above and resuspended in 10 μl water. At this time the two individual samples were pooled and 2 μl of the DNA sample was digested with high activity BglII restriction enzyme (NEB) for 4 hours at 37°C, ethanol precipitated as above and resuspended in 22 μl water. The entire sample was transformed by electroporation into 3 μl E. coli DH10B (Invitrogen) in a 1 mm gap cuvette and recovered in 75 μl S.O.C. media (Invitrogen). 50 μl of sample was immediately plated onto two LB-Ampicillin plates prepared with IPTG and X-gal and incubated at 37°C for 16 hours. White colonies were picked and cultured overnight in 3 ml LB-Ampicillin broth. The DNA was extracted by crude boiling mini-prep and digested with HindIII restriction enzyme (NEB) and run on a 0.8% agarose gel to screen for positive excision events.For the background control 1 μl of the pooled Hirt extracted DNA was resuspended in 21 μl of water and transformed into 3 μl of DH10B E. coli and recovered in 75 μl of S.O.C. media. 5 μl of this preparation was immediately plated on two LB-Ampicillin agar plates containing IPTG and X-gal and incubated at 37°C for 16 hours and the blue colonies were counted.piggyBac antibody5 ml of rabbit anti-serum was obtained from Proteintech Group, Inc. after injection with truncated piggyBac protein. The antiserum was subjected to 50% ammonium sulfate precipitation for 60 minutes at 4°C and centrifuged at 10,000 g for 60 minutes. After resuspending the ammonium sulfate pellet in 20 mM K2HPO4 pH 6.8, 50% ammonium sulfate precipitation was performed again. After centrifugation, the resulting pellet was resuspended again and underwent buffer exchange in 20 mM K2HPO4 using a Centriprep YM-50 (Fisher Scientific, St. Louis, MO) according to manufacturer's protocol. This was followed by concentration of the IgG protein fraction using a Centricon YM-50 (Fisher Scientific). The concentrated IgG fraction was further purified using a 15.5 mL DEAE Affi-Gel Blue Gel Column (Bio-Rad Laboratories, Hercules, CA) according to manufacturer's protocol. The elution buffer was 20 mM K2HPO4, 0.02% sodium azide pH 6.8. The eluted IgG anti-piggyBac antibody was concentrated using a Centricon YM-50 to a final concentration of 16.57 mg/mL as determined by optical density spectrophotometry at 280 nm on a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE). Anti-piggyBac antibody function was verified through western blotting of known positive and negative protein samples (data not shown).Western BlotS2 Cells were co-transfected as described above along with two additional samples containing 1 μg of either a mutant helper, phsp-pBac, as a positive control or a non-piggyBac expression plasmid as a negative control. 48 hours post-induction, the cells were collected and pelleted by centrifugation at 3,000 rpm for 2 minutes. This was followed by two 1 ml washes with 1× PBS. The resulting cell pellets were lysed by resuspending in 100 μl 2× Laemmli Sample Buffer containing 10 mM benzamidine, 10 mM sodium fluoride, 100 mM sodium vanadate, 1 mM phenylmethanesulphonylfluoride (PMSF), 25 μg/ml leupeptin, 25 μg/ml aprotinin, and 25 μg/mL pepstatin. The whole cell lysates were then sonicated on ice for 15 seconds. The protein concentration was measured by optical density spectrophotometry at 280 nm on a Nanodrop ND-1000 spectrophotometer.The whole cell lysates were prepared for electrophoresis on a 1.0 mm thick 7.5% acrylamide SDS-PAGE gel by diluting 125 μg of total protein to a total volume of 25 μl using 2× Laemmli sample buffer. This was followed by heating for 5 minutes at 95°C with 1.25 μl 2-mercaptoethanol to completely denature the protein. The samples were loaded onto two separate gels with 5 samples per gel and electrophoresed at 100V using 1× Tris-glycine plus 0.1% SDS as the running buffer. The protein standards used were Invitrogen's Benchmark Prestained protein ladder. After electrophoresis was complete, the separated proteins were transferred to a Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) using the Bio-Rad Mini Trans-Blot Cell and Module for 1 hour at 350 mAmps. 1× Tris-glycine with 20% methanol was the transfer buffer.Following transfer, the membranes were stained with Ponceau stain and subsequently destained with 5% acetic acid and 1× PBS washes (images not shown). The blots were blocked using 1× PBS plus 5% non fat dry milk for 2 hours while shaking at room temperature. The blots were subsequently probed for piggyBac protein expression using a 1/1000 dilution of anti-piggyBac, purification described above, or for actin using anti-actin I-19 (Santa Cruz Biotechnology, Santa Cruz, CA) in a 1/500 dilution with 1× PBS plus 0.1% Tween-20 (PBS-T) and 5% non-fat milk for one hour while shaking at room temperature. After the primary incubation, the blots were washed four times for five minutes each with 1× PBS-T. The secondary antibody, ECL rabbit IgG, HRP-linked whole antibody (Amersham Biosciences) or donkey anti-goat IgG-HRP (Santa Cruz Biotechnology), respectively, was added in a 1/5000 dilution with 1XPBS-T and 5% non-fat dry milk for one hour at room temperature while shaking. This was followed by washing with 1× PBS-T four times for five minutes each. The blots were developed with Pierce's SuperSignal West Dura Extended Duration Substrate kit (Pierce, Rockford, IL) according to manufacture protocol except 0.5 ml of each substrate was used per blot.Statistical analysisData are expressed as a mean of three replicates +/- standard. ANOVA test was performed using GraphPad Prism 3.0 software. All values were adjusted to a positive control setting of 100%. Means were considered statistically significant when p-values less than 0.05 were obtained with the Dunnett's post-test. Statistical significance is indicated on all data figures as asterisks above bars.Authors' contributionsJHK created the donor plasmids used in the blue/white colony screen, performed all aspects of the blue/white screen, performed the sequence alignment, BoxShade processing and alignment analysis, and prepared the manuscript. CAS performed the western blot and prepared parts of the manuscript dealing with the western blot. TSF performed all aspects of the REN colony screen. MJF conceived of the study and provided guidance.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2533026.json

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+{
+    "id": "PMC2533026",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533026\nAUTHORS: Benjamin Haibe-Kains, Christine Desmedt, Fanny Piette, Marc Buyse, Fatima Cardoso, Laura van't Veer, Martine Piccart, Gianluca Bontempi, Christos Sotiriou\n\nABSTRACT:\nBackgroundDuring the last years, several groups have identified prognostic gene expression signatures with apparently similar performances. However, signatures were never compared on an independent population of untreated breast cancer patients, where risk assessment was computed using the original algorithms and microarray platforms.ResultsWe compared three gene expression signatures, the 70-gene, the 76-gene and the Gene expression Grade Index (GGI) signatures, in terms of predicting distant metastasis free survival (DMFS) for the individual patient. To this end, we used the previously published TRANSBIG independent validation series of node-negative untreated primary breast cancer patients. We observed agreement in prediction for 135 of 198 patients (68%) when considering the three signatures. When comparing the signatures two by two, the agreement in prediction was 71% for the 70- and 76-gene signatures, 76% for the 76-gene signature and the GGI, and 88% for the 70-gene signature and the GGI. The three signatures had similar capabilities of predicting DMFS and added significant prognostic information to that provided by the classical parameters.ConclusionDespite the difference in development of these signatures and the limited overlap in gene identity, they showed similar prognostic performance, adding to the growing evidence that these prognostic signatures are of clinical relevance.\n\nBODY:\nBackgroundDuring the last two decades, several clinical and pathological parameters have been used to evaluate the prognosis of breast cancer (BC) patients. Although different guidelines have been developed to assist clinicians in selecting patients who should receive adjuvant therapy, such as the St Gallen consensus criteria [1], the NIH guidelines [2] or Adjuvant! Online [3], it still remains a challenge to distinguish those patients who would really need adjuvant systemic therapy from those who could be spared such a treatment.With the advent of array-based technology and the sequencing of the human genome, new insights into BC biology and prognosis have emerged. Interestingly, several groups conducted comprehensive genome-wide assessments of gene expression profiling and identified prognostic gene expression signatures [4-6]. To this end, different approaches have been used: 1/ the \"top-down\" (data-driven) approach and 2/ the \"bottom-up\" (hypothesis-driven) approach.Examples of signatures which were developed using the first approach, i.e. by seeking gene expression profiles that are associated or correlated with clinical outcome without any a priori biological assumption, are the 70- and 76-gene signatures developed by the Netherlands Cancer Institute in Amsterdam with Rosetta Informatics-Merck, and the Erasmus MC in Rotterdam together with Veridex, respectively [5,6]. Although these signatures were built using a different microarray platform and had only a small gene overlap, a feature common to both signatures is that they correctly identified the high-risk patients while also identifying a higher number of low-risk patients not needing treatment compared to the clinical guidelines. In order to investigate the enormous potential of these signatures towards better individualization of treatment options in BC therapy, TRANSBIG, a network for translational research established by the Breast International Group (BIG), recently conducted a validation study of the 70-gene and 76-gene signatures which demonstrated the reproducibility and robustness of the 70- and 76-gene signatures [7,8]. This important validation work has led to the implementation of one of the first prospective clinical trials, MINDACT (Microarray In Node-negative Disease may Avoid Chemotherapy Trial) which evaluates the benefit/risk ratio of chemotherapy when the assessment of prognosis based on clinico-pathological features differs from that provided by the 70-gene signature assessed by the MammaPrint™ [9].An example of deriving a prognostic gene expression signature using a hypothesis-driven approach was the study reported by our group that focused on histological grade, a well-established pathological parameter rooted in the cell biology of BC [4]. Indeed, clinicians face a huge problem with respect to patients who have intermediate-grade tumors (grade 2), as these tumors, which represent 30% to 60% of cases, are the major source of inter-observer discrepancy and may display intermediate phenotype and survival, making treatment decisions for these patients a great challenge, with subsequent under- or over-treatment. Performing a supervised analysis, we developed a Gene expression Grade Index (GGI) score based on 97 genes. These genes were mainly involved in cell cycle regulation and proliferation and were consistently differentially expressed between low and high grade breast carcinomas. Interestingly, the GGI was able to reclassify patients with histological grade 2 tumors into two groups with distinct clinical outcomes similar to those of histological grade 1 and 3, respectively.In addition to the signatures described above, many other research groups have contributed gene expression signatures that are predictive of clinical outcome in BC [10]. However, given the fact that their performances were evaluated on different datasets with limited or no independent validation and that there is only little gene overlap between the different gene sets, which can be attributed to the different platforms, training sets, and/or statistical tools used, it is unclear which is the best. The public availability of the TRANSBIG series gives the opportunity to perform a thorough comparison of several gene signatures. Indeed, this dataset of untreated primary BC patients is the only one on which three gene signatures, the 70-gene, the 76-gene and the GGI signatures, were computed using the original algorithms and microarray platforms [7,8], providing also the advantage that this population was not used for the development of any of these signatures. Here, we statistically compared these three signatures in terms of predicting clinical outcome for the individual patient using two performance criteria.MethodsGene expression and clinical dataGene expression and clinical data of TRANSBIG series [7,8] were retrieved from EMBL-EBI ArrayExpress (, accession number E-TABM-77) and NCBI GEO (, accession number GSE7390) databases, for the validation of the 70-gene signature (TBAGD) and of the 76-gene signature (TBVDX), respectively. The original TRANSBIG series included 309 patients for whom the 70-gene signature was computed using the Agendia clinical MammaPrint™ 1.9 k Agilent custom microarray chip. This series is referred to as TBAGD. In a second time, the 76-gene signature was computed for a subset of these patients for whom there was enough material left. The Affymetrix HG-U133A research GeneChip™ was used for the signature computation. This series of 198 patients is referred to as TBVDX. Finally, we were able to compute the gene expression grade index in TBVDX as this series used Affymetrix technology. In this paper, we used the TBVDX patient's series for which we had the official classification for the three gene signatures, i.e. the 70-gene, 76-gene and the gene expression grade index.Risk statusWe considered only the binary risk status for the survival analysis, as the continuous risk scores are not publicly available for 70- and 76-gene signatures in the TBAGD and TBVDX series, respectively. The TBAGD series is composed of 307 BC patients and the TBVDX series is composed of 198 BC patients who are also included in the TBAGD series. In order to use the GGI as a prognostic signature, we first identified a threshold that allows to define the binary risk status according to the GGI scores, on the dataset of 286 patients used by Wang et al. (VDX, [6]). Indeed, the threshold used in the original publication [4] was selected to optimize the discrimination between patients with histological grade 1 and 3 tumors. As this threshold was not suited for survival analysis, we used the same training set as the 76-gene signature to keep the TRANSBIG series fully independent. We did not attempt to select a threshold optimizing some performance criteria, e.g. hazard ratio or logrank p-value, in order to avoid overfitting in VDX. Instead, we selected a threshold based on tertiles (the third of the patients having the lowest GGI scores being defined as low-risk and the remaining patients as high-risk) leading to similar repartition of patients in low- and high-risk groups to the 76-gene signature. The GGI score was computed as in Sotiriou et al. [4] except that we performed a robust scaling instead of the original scaling method to avoid the use of histological grade information. After the robust scaling, the GGI scores have an interquartile range equals 1 and a median equals 0 within the dataset. The risk status computed using the threshold based on tertiles, yielded good classification performance on the VDX dataset (HR of 2.12; 95% CI: 1.35–3.34; p = 0.001). The GGI continuous risk scores for TBVDX were computed as for VDX and the GGI binary risk status was defined using the threshold identified on VDX. The clinical risk status was defined using the Adjuvant! Online software (AOL, ) as in the validation study conducted by the TRANSBIG consortium [7,8].Classification associationWe used Cramer's V statistic [11] to quantify the strength of the association between two gene signature classifications. The values range from 0 to 1, with 0 indicating no association and 1 indicating a perfect association. Traditionally, values of 0.36 to 0.49 indicate a substantial association, and values of 0.50 or more indicate a strong association. The significance of such an association was computed using a chi-squared test.Survival analysisWe considered the distant metastasis free survival (DMFS), time to distant metastasis (TDM) and overall survival (OS) of BC patients as the survival endpoints. We performed the survival analyses by censoring the survival data at 10 years and by considering the full follow-up. We show the results for DMFS censored at 10 years in the article. The results for TDM and OS censored at 10 years are reported in [Additional File 1]. The survival analyses with the full follow-up are also reported in [Additional File 1]. Sensitivity and specificity were estimated at 3, 5 (the endpoint used to derive the 70- and 76-gene signatures), 10 and 15 years and by considering the full follow-up as well. Sensitivity was defined as the probability that a patient who experienced the event of interest was in the high-risk group and specificity as the probability that a patient who did not experience the event of interest was in the low-risk group. We used the nearest neighbors estimator defined in [12] in order to take into account the time of events and the censoring. Hazard ratios between two groups were estimated through univariate Cox's proportional hazard regression models, stratified by clinical center to account for the possible heterogeneity in patient selection or other potential confounders among the various centers. Hazard ratios for the risk groups defined by the gene signature were also estimated with stratification for clinical risk in order to reflect the prognostic impact of the gene signature over and above that of clinicopathological variables (\"adjusted hazard ratios\"), as reported previously in [7,8]. In addition to the hazard ratio, we used the concordance index to quantify the predictive ability of a survival model [13]. It estimates the probability that, of a pair of randomly chosen comparable patients, the patient in the high-risk group will recur before the patient in the low-risk group. A pair of patients is comparable if one of the patients recurred before the other patient and if the patients are in different risk groups. Standard error for the concordance index was estimated based on the asymptotic normality of its estimate [14]. The difference in hazard ratios and concordance indices were computed using a paired Student t test. Survival curves were computed through the Kaplan-Meier product limit estimator and their difference was tested in a univariate Cox model, stratified when required.All p-values were two-tailed and p-values < 0.05 were considered statistically significant. All statistical analyses were carried out using R version 2.5.1 [15].ResultsRisk status computed by the prognostic signaturesWe used the original algorithms and microarray platforms to compute the risk status of 198 patients used in the second TRANSBIG validation study [8]. Similarly to the GENE70 [5] and GENE76 [6] signatures, we performed a calibration step in order to compute GGI classification in this independent series. This step makes the prediction of a single patient challenging, as it requires a large number of samples. However, the standardization of hybridization protocols, the setup of a central laboratory to carry out the microarray experiments and the use of test samples to calibrate the process might help to avoid this issue. The MAQC consortium [16] is specifically studying this problem in order to bring the microarray-based gene signatures into clinic.Patient characteristics according to the prognostic signaturesThe patients were younger than the age of 61 (median age 47), had node-negative, T1–T2 (≤ 5 cm) tumors and did not receive any adjuvant treatment. The tumor samples from these patients were previously hybridized on the Agilent platform to define the 70-gene signature [6,17], as well as on the Affymetrix platform, from which the 76-gene signature [6] and Gene expression Grade Index (GGI, [4]) were computed. Patient characteristics are shown in Table 1, organized according to their genomic risk of recurrence as defined by the 70-gene and the 76-gene signatures as well as by the GGI, and by their clinical risk as defined by Adjuvant! Online (AOL, [3,7,8]). The distribution of the risk categories was similar for the different signatures in terms of patient's age and tumor size. However, differences in risk distribution were observed between the 76-gene signature and the two others in terms of tumor grade and estrogen receptor (ER) status. Indeed, the 76-gene signature identified a higher proportion of high-risk grade 2 tumors and low-risk grade 3 tumors, high-risk ER-positive and low-risk ER-negative tumors. When looking at the distribution of the high and low-risk patients according to the ER status, it appears clearly that these signatures mainly impact on the prediction of clinical outcome on ER-positive patients. Compared to the different genomic risk classifications, the clinical risk classification (AOL) identifies a higher proportion of high-risk patients in the older subgroup or in the group of patients with large tumors. None of the patients whose tumors were moderately/poorly differentiated or ER-negative are considered as low-risk by AOL.Table 1Characteristics of patients of the TRANSBIG validation series (n = 198), according to the 70-gene signature (GENE70), the 76-gene signature (GENE76), the Gene expression Grade Index (GGI) and the Adjuvant! Online (AOL) risk classifications.SignatureGENE70GENE76GGIAOLNumber of patientsLow-risk (N = 66)High-risk (N = 132)Low-risk (N = 55)High-risk (N = 143)Low-risk (N = 69)High-risk (N = 129)Low-risk (N = 46)High-risk (N = 152)Age < 41 years113110321032438 41–50 years3367247633673367 51–60 years333424352630947Size T1ab (< 1 cm)45454581 T1c (1–2 cm)2435174225342138 T2 (2–5 cm)38923496409017113Tumor grade Good differentiation171314162010237 Intermediate4241216244392360 Poor differentiation7762063578083 Unknown02020202Estrogen receptors Positive6371419365694688 Negative3611450460064Concordance of classification of samplesFigure 1 illustrates the classification of the tumor samples according to the prognostic signatures. We observed agreement in prediction in 135 of 198 patients (68%) when considering the three signatures. When comparing the signatures two by two, agreement in prediction was 71% for the 70- and 76-gene signatures, 76% for the 76-gene signature and the GGI, and 88% for the 70-gene signature and the GGI. The strength of the concordance of classifications, quantified through Cramer's V statistic, was 0.33 for the 70- and 76-gene signatures, 0.47 for the 76-gene signature and the GGI, and 0.76 for the 70-gene signature and the GGI.Figure 1Venn diagram illustrating the classification of the tumor sample according to the prognostic signatures. Dark red = high-risk patients and blue = low-risk patients. GENE70 = 70-gene signature, GENE76 = 76-gene signature, and GGI = Gene expression Grade Index.Survival analysesIn this section, we report the results from the survival analyses using the DMFS censored at 10 years and with the full follow-up. We performed these two separate analyses in order to highlight the time-dependency of the gene signatures as shown in [7,8].Survival data censored at 10 yearsTo assess the prognostic ability of the three signatures, we first compared their concordance index, which is used to quantify the predictive ability of a survival model. Although all three concordance indices were highly significant, the 70-gene signature and GGI displayed a higher concordance index compared to the 76-gene signature (0.90 compared to 0.80; Figure 2A). However, this difference was not statistically significant (Table 2). In contrast, the clinical risk calculated using AOL displayed a lower concordance index value (0.69) compared to either ones generated by the genomic signatures.Table 2P-values of the Student t test for the difference between concordance indices and hazard ratios for the 70-gene signature (GENE70), the 76-gene signature (GENE76), and the Gene expression Grade Index (GGI) risk classifications.p-value for difference in concordance indicesp-value for difference in hazard ratiosGENE70 vs GENE760.150.11GENE70 vs GGI0.530.42GENE76 vs GGI0.220.19Figure 2Forest plots (and 95% CI) for the three gene signatures and the Adjuvant! Online classification showing: A/the concordance indices, and B/the log2 hazard ratios. GENE70 = 70-gene signature, GENE76 = 76-gene signature, GGI = Gene expression Grade Index and AOL = Adjuvant! Online.We next performed univariate and multivariate Cox analyses, which included the traditional clinico-pathological parameters, for each signature separately. The univariate hazard ratios (HR) were 7.12 (95% CI: 2.52–20.11; p = 2.1 × 10-4), 3.18 (95% CI: 1.35–7.53; p = 8.4 – 10-3) and 5.85 (95% CI: 2.3–15; p = 2.1 – 10-4) for the 70-gene signature, 76-gene signature and the GGI respectively. We additionally computed the HR for the clinical risk as defined by AOL, which was not statistically significant for DMFS evaluation in this cohort of patients (2.01; 95% CI: 0.89–4.5; p = 0.091). The log2 of these HR are illustrated in Figure 2B. Although the HR of the 70-gene signature and the GGI were higher than the HR of the 76-gene signature, the differences were not statistically significant (see Table 2). Figure 3 illustrates the Kaplan-Meier estimates of DMFS for the four groups of patients (two groups with concordant results in risk assessment and two with discordant results) for the different signatures two by two.Figure 3Kaplan-Meier curves for distant metastasis free survival for: A/the 70-gene signature vs the 76-gene signature; B/the 70-gene signature vs the Gene expression Grade Index, and C/the 76-gene signature vs the Gene expression Grade Index. GENE70 = 70-gene signature, GENE76 = 76-gene signature and GGI = Gene expression Grade Index.From the multivariate analyses (Table 3), we can conclude that the three signatures added significant information to the traditional parameters and were the strongest predictive variables of DMFS, as reflected by their lowest p-values compared to the other variables. The additional information of these signatures over the clinical risk was also confirmed by the fact that the univariate HRs for the three signatures remained similar when adjusted for the clinical risk, with a HR of 7.25 (95% CI: 2.4–21.5; p = 3.5 – 10-4), 2.8 (95% CI: 1.2–6.8; p = 0.018) and 6.25 (95% CI: 2.3–17; p = 3.3 – 10-4) for the 70-gene signature, 76-gene signature and the GGI respectively.Table 3Multivariate Cox analyses for the 70-gene signature (GENE70), the 76-gene signature (GENE76) and the Gene expression Grade Index (GGI) risk classifications.GENE70GENE76GGIHR (95% CI)p-valueHR (95% CI)p-valueHR (95% CI)p-valueAge (≤ or >50 years)1.51 (0.82–2.79)0.31.78 (0.97–3.25)0.0621.73 (0.94–3.16)0.077Tumor Size (≤ or >2 cm)1.3 (0.72–2.5)0.361.27 (0.68–2.37)0.451.22 (0.65–2.29)0.53ER status0.82 (0.43–1.6)0.570.6 (0.31–1.17)0.130.78 (0.4–1.51)0.46Grade0.93 (0.34–2.53)0.891.51 (0.53–4.28)0.50.75 (0.27–2.08)0.58Risk according to the gene signature7.1 (2.4–21)4 × 10-43.39 (1.41–8.12)6 × 10-36.42 (2.36–17.45)3 × 10-4Lastly, we combined the three gene signatures in order to assess the potential improvement in BC prognostication. We used a simple combination scheme that defined the risk of a patient as the sum of the classifications (low-risk = 0 and high-risk = 1) by the three gene signatures. As illustrated in Supplementary Figure 1 in [Additional File 1], the patients for whom the three gene signature classifications were concordant are well defined, with only 2 patients relapsing in the low-risk group after 9 years of follow up. However, the patients with discordant classifications exhibited good survival and their survival curves were not distinguishable. The combination of the three gene signatures did not yield significant improvement in prognostication (the hazard ratio between the concordant cases, i.e. 'All Low' and 'All High', is not significantly higher than when each gene signature was considered separately), maybe due to their high concordance and the sample size of the TBVDX seriesSurvival data with the full follow-upWe computed the concordance index of all the gene signatures using the survival data with the full follow-up. The three concordance indices were significant. We observed higher concordance indices for the 70-gene signature and GGI compared to the 76-gene signature (0.84 and 0.79 for GENE70 and GGI respectively compared to 0.71 for GENE76; Supplementary Figure 12 in [Additional File 1]). This difference was not statistically significant (Supplementary Table 5 in [Additional File 1]) although we noted a trend for GENE70 to have a higher concordance index (p = 0.065). In contrast, the clinical risk calculated using AOL displayed a lower concordance index value (0.69) compared to either ones generated by the genomic signatures.We next performed univariate and multivariate Cox analyses, which included the traditional clinico-pathological parameters, for each signature separately. The univariate hazard ratios (HR) were 2.77 (95% CI: 1.41–5.43; p = 3.1 – 10-3), 1.76 (95% CI: 0.92–3.34; p = 0.086) and 2.41 (95% CI: 1.29–4.5; p = 5.9 – 10-3) for the 70-gene signature, 76-gene signature and the GGI respectively. We additionally computed the HR for the clinical risk as defined by AOL, which was not statistically significant for DMFS evaluation in this cohort of patients (1.5; 95% CI: 1.29–4.5; p = 0.11). The log2 of these HR are illustrated in Supplementary Figure 13 in [Additional File 1]. Although the HR of the 70-gene signature and the GGI were higher than the HR of the 76-gene signature, the differences were not statistically significant (see Supplementary Table 5 in [Additional File 1]). Supplementary Figures 14–16 in [Additional File 1] illustrate the Kaplan-Meier estimates of DMFS for the four groups of patients for the different signatures two by two.From the multivariate analyses (Supplementary Table 6 in [Additional File 1]), we can conclude that the three signatures added significant information to the traditional parameters and were the strongest predictive variables of DMFS, as reflected by their lowest p-values compared to the other variables. We computed the univariate HRs adjusted for the clinical risk, i.e. 2.8 (95% CI: 1.35–5.82; p = 5.8 – 10-3), 1.55 (95% CI: 0.81–2.97; p = 0.18) and 2.13 (95% CI: 1.12–4.02; p = 0.02) for the 70-gene signature, 76-gene signature and the GGI respectively.Contrary to the analyses using the survival data censored at 10 years, the HRs with and without adjustment for clinical risk were not significant for GENE76, highlighting the decrease in performance we observed by using the survival data with the full follow-up. This performance degradation was due to a group of late relapses occurring after 10 years of follow-up, classified as low-risk by the three gene signatures (see Supplementary Figure 17 in [Additional File 1]).We combined the three gene signatures using the method described previously. In agreement with the results from survival data censored at 10 years, the combination did not yield significant improvement in prognostication (see Section 2.1.1 in [Additional File 1]).Sensitivity and specificityWe computed the sensitivity and the specificity for DMFS at 5 years for the three signatures as well as for the clinical risk as defined by AOL. These signatures exhibited high sensitivities (0.97 to 1) compared to the clinical risk (0.88). Similarly to the results reported in previous publications [5-8,18,19], the gene signatures exhibited low specificities (0.33 to 0.42) which were however higher than the specificity associated with clinical risk assessment (0.26). The estimations of sensitivity and specificity for DMFS, TDM and OS at 3, 5, 10, and 15 years and by considering the full follow-up, are given in Supplementary Tables 7, 10 and 13 [see Additional File 1]. Although the gene signatures yielded higher sensitivities and specificities than clinical risk until 10 years, we observed a decrease in performance with increasing follow-up duration (10 years and more). The specificities of the gene signatures remained higher than clinical risk but their sensitivities were slightly lower.The results were highly concordant between the survival endpoints, namely DMFS, TDM and OS [see Additional File 1].Discussion and conclusionThe objective of this study was to conduct an unbiased comparison of three different prognostic signatures. To this end, the signatures had to be evaluated on their original platform and computed with their original algorithms on an independent population of untreated BC patients. All these requirements were met by the TRANSBIG validation series [7,8]. The results showed that the three evaluated signatures had similar capabilities of predicting DMFS (TDM and OS [see Additional File 1]) in this set of patients and added significant prognostic information to that provided by the classical parameters.Two groups recently undertook to compare different prognostic signatures. Fan et al. reported that the intrinsic subtypes and several prognostic signatures [5,6,20-24] gave similar outcome predictions for the individual patient when investigated on a single dataset [25]. Although this study yielded promising conclusions, some issues remained open: 1/ the dataset which was considered for this study was used for the development of some gene sets and could then not be considered as a true independent validation set for all the evaluated signatures and 2/ since some of these signatures were developed on another platform, the initial algorithms could not be applied. Thomassen et al. compared nine prognostic signatures in a cohort of low-malignancy BC patients [26]. In their study, they also compared the same signatures as we did, but computed the associated risk classification from data generated on a different platform, with as consequence that not all the genes from the 76-gene signature and the GGI were represented and that they could not use the original algorithms. Although the proportions of samples that they reported with identical classification were slightly higher to ours, the rank of concordances was similar with 83% between the 70- and 76-gene signatures, 85% between the 76-gene signature and the GGI, and 92% (the highest agreement) between the 70-gene signature and the GGI.Thanks to the long follow-up of the TRANSBIG series (up to 25 years), we were able to assess the performance of the three gene signatures with respect to the time. In agreement with Buyse et al. [7] and Desmedt et al. [8], we observed a strong time dependence of the classification performance. The gene signatures yielded good performance at 10 years but we observed a strong degradation when considering the full follow-up due to the poor identification of late relapses (after 10 years). That might be due to: (i) the biology, Klein et al. have suggested that the biological phenomenon responsible for the appearance of early and late relapses might be different [27,28]; (ii) the statistical method, the GENE70 and GENE76 signatures have been developed to predict early relapses (distant metastasis before 5 years) as in the original publications, the authors controlled the sensitivity and the specificity of these signatures for early relapses only; (iii) the quality of survival data, although it is hard to quantify, one could intuitively think that the quality of survival data decreases with respect to the duration of follow-up since it is difficult to follow patients during a long period (high level of censoring).While there are only partial or very small to no overlaps between the different prognostic gene signatures [29], there is still a relatively high agreement of classification of the patients between the different signatures. We may assume that these similar outcome predictions are based on representation of largely overlapping biological processes. This is supported by several reports. Indeed, Thomassen et al. found that cell cycle and cell proliferation represented the predominant overlaps in gene ontology categories of the nine prognostic signatures they compared [26]. Yu et al. also conducted pathway analyses of five published prognostic gene signatures and also found that the signatures had many pathways in common such as cell cycle, regulation of cell cycle, mitosis, apoptosis, etc [29]. Our group also investigated in a large meta-analysis of publicly available gene expression data extensive analysis how different gene lists may give rise to signatures with equivalent prognostic performance and found by dissecting these signatures according to the main molecular processes involved in BC, that proliferation may be the common driving force of several prognostic signatures [30]. This might explain why the combination of the three gene signatures evaluated in this study did not yield significant improvement in prognostication.Until now, the generation of the prognostic signatures has been done on global sets of BC patients. However, since it is clear that BC is a molecular heterogeneous disease, with subgroups defined primarily by the estrogen (ER) and HER2 receptors, prognosis could be refined to these molecularly homogeneous subgroups of patients. We showed for example in our meta-analysis that proliferation is the strongest parameter predicting clinical outcome in the ER+/HER2- subgroup of patients only, whereas immune response and tumor invasion appear to be the main biological processes associated with prognosis in the ER-/HER2- and HER2+ subgroups respectively [30]. This could also have implications with regard to the evaluation of response to different therapies [31] and help to define new therapeutic strategies in the specific molecular subgroups of BC patients.To conclude, our study showed that although prognostic signatures may have been developed using a different approach, different platforms and statistical tools on different sets of comparable patients, with a small overlap in gene identity as a consequence, they can result in similar predictions of outcome. Although the technology used has been shown to be ready for clinical practice [16], and can be used as one parameter in combination with current clinical parameters, these signatures need to be prospectively validated to prove their superiority and benefit above and beyond the use of standard clinico-pathological prognosis variables to guide the choice of adjuvant therapy. Two gene signatures, the 70-gene signature which has been studied in this paper, and the recurrence score [21] have reached the final step of prospective testing in the MINDACT (Microarray in Node Negative Disease May Avoid Chemotherapy) and TAILORx trials, respectively. We believe that the results from these studies will help to guide future BC treatment.Competing interestsChristos Sotiriou, Mauro Delorenzi and Martine Piccart are named inventors on a patent application for the Genomic Grade signature used in this study. Laura van't Veer is founder and stock owner of Agendia. There are no other conflicts of interest.Authors' contributionsBH–K, CD, CS were responsible for the design and execution of the study, data and statistical analysis and interpretation, and final manuscript writing; FP, MB, GB, FC, LV, MP supported the data and statistical analysis and interpretation; GB, CS supervised the study. MP, CS provided the study funding. All authors read and approved the final manuscript.Supplementary MaterialAdditional file 1Supplementary information.Click here for file\n\nREFERENCES:\nNo References"
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+    "id": "PMC2533028",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533028\nAUTHORS: Arnab Bhattacharya, Sasha Levy, Adria LeBoeuf, Michelle Gaylord, Leslie Wilson, Ambuj K Singh, Stuart C Feinstein\n\nABSTRACT:\nBackgroundInnumerable biological investigations require comparing collections of molecules, cells or organisms to one another with respect to one or more of their properties. Almost all of these comparisons are performed manually, which can be susceptible to inadvertent bias as well as miss subtle effects. The development and application of computer-assisted analytical and interpretive tools could help address these issues and thereby dramatically improve these investigations.ResultsWe have developed novel computer-assisted analytical and interpretive tools and applied them to recent studies examining the ability of 3-repeat and 4-repeat tau to regulate the dynamic behavior of microtubules in vitro. More specifically, we have developed an automated and objective method to define growth, shortening and attenuation events from real time videos of dynamic microtubules, and demonstrated its validity by comparing it to manually assessed data. Additionally, we have used the same data to develop a general strategy of building different models of interest, computing appropriate dissimilarity functions to compare them, and embedding them on a two-dimensional plot for visualization and easy comparison. Application of these methods to assess microtubule growth rates and growth rate distributions established the validity of the embedding procedure and revealed non-linearity in the relationship between the tau:tubulin molar ratio and growth rate distribution.ConclusionThis work addresses the need of the biological community for rigorously quantitative and generally applicable computational tools for comparative studies. The two-dimensional embedding method retains the inherent structure of the data, and yet markedly simplifies comparison between models and parameters of different samples. Most notably, even in cases where numerous parameters exist by which to compare the different samples, our embedding procedure provides a generally applicable computational strategy to detect subtle relationships between different molecules or conditions that might otherwise escape manual analyses.\n\nBODY:\nBackgroundStatement of ProblemInnumerable biological investigations require comparing different members of a collection of entities with respect to one or more properties. The conclusions to be drawn from such studies are based on an analysis of the degree of similarity or dissimilarity among the different members. For example, one might compare the activity of different isoforms or fragments of a protein of interest, or compare wild type protein(s) with various mutant versions of a protein that causes a disease state. Many additional examples come from comparisons of data sets derived from microarray and proteomics studies, as well as population genetics. Given the technical advances of recombinant DNA technology and the explosion in genomics over the past few years, it is a certainty that the number of these sorts of comparative studies, and the number of entities to be compared within each study, will increase dramatically in the near future. Unfortunately, the vast majority of such comparative studies are currently performed manually, with investigators searching for similarities and dissimilarities among different test entities \"by eye\". This is especially difficult when each member of the collection is being characterized by multiple criteria. The analytical process is time consuming, likely to miss subtleties and is susceptible to inadvertent bias and human errors. Development and application of computer-assisted modeling and visualization can provide extraordinarily valuable data analyses and interpretive tools for assessing relationships among different members in a study.Microtubules and Microtubule DynamicsMicrotubules represent one of the three main components of the eukaryotic cellular cytoskeleton [1]. They are hollow, unbranched cylinders, formed by the non-covalent association of αβ tubulin dimer subunits. Microtubules serve a wide variety of essential structural and transport functions, including the segregation of chromosomes during cell division and the transport of vesicular cargo up and down long axonal processes in neurons.Microtubules are highly dynamic structures, gaining and losing tubulin dimer subunits by a stochastic process known as dynamic instability [2,3]. A large body of data, both pharmacological and somatic cell genetics, has led to the conclusion that proper regulation of microtubule dynamics is essential in order for microtubules to perform their many critical cellular functions (for review, see [4]). For example, the effectiveness of the anti-cancer drug taxol derives from its ability to suppress microtubule dynamics, thereby interfering with the ability of cancer cells to proliferate [5]. Given the importance of properly regulated microtubule dynamics, it is not surprising that cells have evolved a host of regulatory proteins that finely tune microtubule dynamics, including tau, MAP2, MAP4, SCG10 and stathmin.The Microtubule Associated Protein TauThe microtubule associated protein tau is essential for the normal development and maintenance of the nervous system [6-8]. It binds directly to microtubules [9,10], and its ability to regulate microtubule dynamics [11-13] is itself tightly regulated by both alternative RNA splicing [14] and phosphorylation (for review, see [15]). Alternative RNA splicing leads to the synthesis of two classes of tau, known as 3-repeat tau and 4-repeat tau (See Figure 1 for a schematic). Whereas normal human fetal brain expresses only 3-repeat tau, adult human brain expresses approximately equal amounts of 3-repeat and 4-repeat tau. Despite this dramatic developmental shift in expression profiles, the functional and mechanistic differences between 3-repeat and 4-repeat tau remain poorly understood. While it is well-established that 4-repeat tau is a more potent regulator of microtubule dynamics than 3-repeat tau, there have been indications over the years that the two classes of tau isoforms may also have inherent qualitative differences as well [12,16-19].Figure 1Isoforms of tau. Schematic of two classes of tau isoforms – 4-repeat tau and 3-repeat tau. In 3-repeat tau, the region between repeats 1 and 2 and the second repeat structures are missing by virtue of alternative RNA splicing.Abnormal tau action has long been correlated with neurodegeneration. Indeed, the classic neurofibrillary tangle pathology of Alzheimers and many related dementias are composed primarily of aberrant tau (for example, see [20]). In 1998, a direct cause and effect relationship between errors in tau action and/or regulation and neurodegeneration was established by the genetic linkage between mutations in the tau gene and FTDP-17, a fronto-temporal dementia with many similarities to Alzheimers disease [21-23]. Two classes of tau mutations have been described. The first collection of mutations are structural in nature, caused by various amino acid substitutions in tau. The second class of mutations are especially subtle and provocative – they are caused by errors in alternative tau RNA splicing that alter the expression ratio of otherwise normal 4-repeat and 3-repeat tau molecules. Specifically, rather than a ~50/50 ratio in adult human brain, the mutant ratio is closer to ~75/25. In both the structural and regulatory mutations, the result is early onset of neuronal cell death and dementia.Unfortunately, the molecular mechanisms underlying tau-mediated neuronal cell death remain unclear. One widely held model suggests that errors in tau action lead to the aggregation of tau into neurofibrillary tangles, which are in turn cytotoxic [24]. An alternative model suggests that tau-mediated neuronal cell death results from the inability of tau to properly maintain microtubule dynamics within a narrow range of activities required for cell viability [4,13,19,25]. Additional models have also been proposed (see ).Computational PerspectivesTo quantitatively investigate the regulation of microtubule dynamics under varying conditions (for example, with different tau isoforms or tau:tubulin molar ratios), cell biologists employ video microscopy to visualize and record images of dynamic microtubules in real time. For each condition being assessed, many different individual microtubules must be imaged, tracked and analyzed [19]. From the resulting microtubule \"life history plots\" (Figure 2), the dynamic behaviors of similarly treated microtubules can be determined, such as average growth or shortening rates. Subsequently, the behavior of microtubules under different conditions can be compared.Figure 2Example life history plot. An example of a typical \"life history\" plot of a microtubule, i.e., microtubule length as a function of time plot. The microtubule shown here is from the 3-repeat tau sample at a tau:tubulin molar ratio of 1:38.Computer-assisted methods are especially attractive for time series investigations of this sort. In the specific case of analyzing the regulation of microtubule dynamics, inadvertent bias and non-reproducibility in data interpretation among different labs and different investigators when defining the beginning and end points of individual growth, shortening or attenuation events could become significant. Despite the fact that these events are explicitly defined, investigators must make many judgment calls. In contrast, computer-assisted methods offer a faster and more objective assessment of the data. More importantly, these methods can also provide analytical tools as much as determine the fit of the data to various statistical models, thereby testing various conceptual representations of the underlying molecular mechanisms of action of the system under study. Modeling can also generate testable mechanistic predictions for subsequent investigations. In a general sense, sophisticated computational tools have the potential to make major contributions to many areas of biological research.ResultsThe main goal of this work is to develop general computational tools to quantitatively assess the differences among samples of interest and to visualize those differences in a manner that facilitates their comparison. The data being analyzed is derived from an earlier work in which video microscopy was used to visualize and assess the abilities of 3-repeat and 4-repeat tau to regulate various parameters of microtubule dynamics in in vitro reactions (Levy et al., 2005) [19]. Samples contained purified tubulin dimers and purified recombinant human tau. The two primary variables were (i) the presence of 3-repeat tau, 4-repeat tau or no tau, and (ii) the molar ratio of tau to tubulin. In vivo, the molar ratio of tau:tubulin varies from cell to cell. Further, the ratio can vary even among different regions within single cells, such as cell body versus axon versus growth cone. The different ratios examined are likely to span the range of biologically meaningful values [26].Automated Life History Plot AnalysisWe first developed an automated method to identify the different events – growth, shortening, and attenuation (\"pause\") – on microtubule life-history plots, using a set of pre-defined rules (see the Methods section for details). We then compared the ability of this automated method to determine average microtubule growth rates with standard manual analysis, using data sets assessing the ability of tau to regulate microtubule dynamics from [19]. In this earlier work, microtubule dynamics were assayed under nine different experimental conditions. As seen in Table 1, the deviation between automatic and manually determined rates ranges from 3.33% to 12.68%, with an average deviation of 6.59%. Statistically, the differences between the manually determined values and the automatically measured values are not significant (as shown by the p-values), except for one condition. The p-value is computed by performing a t-test of the manually determined growth rates against the automatically computed ones for each condition (details are in the Methods section). More importantly, the relative order of the conditions do not change, and the degrees of separation are well maintained. Table 2 shows the comparison for the same set of conditions using a different tubulin preparation from [19]. Again, our automated method accurately recapitulates manual analysis with increased objectivity. Additionally, it markedly reduces the time required to conduct these investigations.Table 1Growth rates of tau conditions (sample 1)ConditionManualAutomaticDifferenceDeviationP-value3R-1:203.994.190.204.93%0.153R-1:383.583.760.184.80%0.053R-1:452.022.270.2512.58%0.053R-1:552.022.280.2612.68%0.034R-1:204.714.560.153.33%0.854R-1:383.964.190.235.86%0.154R-1:453.513.650.144.14%0.234R-1:552.592.840.259.45%0.05No-Tau2.302.530.2310.13%0.27Average0.216.59%The manual data is reproduced from Table III of (Levy et al., 2005) [19]. The third column shows growth rates (in μm/min) automatically computed from the manually tracked tips of the microtubules using an objective set of rules with no human interference. The relative ranks of the conditions remain the same.Table 2Growth rates of tau conditions (sample 2)ConditionManualAutomaticDifferenceDeviationP-value3R-1:203.904.020.123.07%0.223R-1:382.672.870.207.49%0.733R-1:452.162.390.2310.65%0.133R-1:552.342.470.135.56%0.354R-1:204.934.990.061.22%0.634R-1:384.394.630.245.47%0.434R-1:453.873.870.000.00%0.754R-1:553.253.470.226.77%0.14No-Tau2.772.950.186.50%0.36Average0.154.55%The manual data is reproduced from Table II of (Levy et al., 2005) [19]. The third column shows growth rates (in μm/min) automatically computed from the manually tracked tips of the microtubules using an objective set of rules with no human interference. The relative ranks of the conditions remain the same.It is also important to note that inherent biological variability exists in the microtubule growth rate data. This likely results from biochemical variations between different tubulin preparations, such as different tubulin isoform expression ratios and/or varying degrees of post-translational modifications, such as phosphorylation, acetylation or tyrosination. For example, assuming each growth rate determination is within a variation of ± 0.1 μm/min, the rank order of the conditions for each of the two data sets is quite similar, although the 4R-1:55 and 3R-1:38 conditions are reversed (see Table 3). This inherent biological variability could limit the utility of some sophisticated and highly sensitive statistical models to make testable predictions regarding mechanisms underlying the regulation of microtubule dynamics. At the minimum, multiple data sets might be necessary and all predictions would need to be considered tentative until tested directly by other biological means.Table 3Rank order of different conditionsRankCondition (growth rate)Table III of (Levy et al., 2005) [19] (Table 1 of this paper)Table II of (Levy et al., 2005) [19] (Table 2 of this paper)14R-1:20 (4.7)4R-1:20 (4.9)24R-1:38 (4.0)4R-1:38 (4.4)33R-1:20 (4.0)3R-1:20 (3.9)44R-1:45 (3.5)4R-1:45 (3.9)53R-1:38 (3.6)4R-1:55 (3.3)64R-1:55 (2.6)3R-1:38 (2.7)7No-Tau (2.3)No-Tau (2.8)83R-1:45 (2.0)3R-1:45 (2.2)93R-1:55 (2.0)3R-1:55 (2.3)The ranks of the conditions remain almost the same across the two samples (considering a variation of 0.1 μm/min) except that the 4R-1:55 and the 3R-1:38 conditions are reversed.Modeling and Embedding StrategyNext, we sought to develop mathematical and statistical models to capture different dynamic aspects of microtubule behavior and to embed them in a two-dimensional space for visualization and easy comparison of different conditions. We used the Sammon projection method [27] for embedding and visualization. In short, the embedding process displays each experimental condition with an (x, y) position; the relative distance between the (x, y) positions of any pair of experimental conditions corresponds to their relative degree of relatedness (details are in the Methods section). The conditions of interest can be compared based on numerous parameters and the computational method is applicable to all kinds of numerical parameters.The outline of our method is as follows. First, the experimental measurements are analyzed based on an appropriate mathematical model. Then, an appropriate dissimilarity function is applied to calculate the relative distances between the models of each pair of conditions. Finally, the conditions are embedded on a two-dimensional space such that the inherent structure of the data is approximately preserved. This is achieved by assigning points (x and y coordinates) to the models such that the Euclidean distance between any pair of points in this space is as close to the original dissimilarity measure between their models as possible. Unlike principal component analysis (PCA) [28], this method works with any distance matrix. The quality of the embedding is measured by distortion. For ideal embeddings, where all dissimilarity values are maintained exactly as Euclidean distances in the embedded space, the distortion is 1. The details of the models, the dissimilarity functions, the embedding algorithm, and the distortion computations are presented in the Methods section.Two-Dimensional Embedding AnalysisMicrotubule Growth RateAs a proof-of-principle exercise, we used the automatically measured values from Table 1 and applied our embedding strategy to compare the abilities of each tau isoform to regulate the growth rate of microtubules. Since these growth rate data are one-dimensional, the distortion is 1, and the embedding procedure should yield a straight line. Figure 3 shows the two-dimensional embedding of the conditions. The requirement for the distances are fulfilled and the points are on a straight line. Additionally, consistent with [19], we observe that very low ratios of 3-repeat and 4-repeat tau:tubulin have opposite effects on the dynamic behavior of microtubules. More specifically, while 1:55 and 1:45 3-repeat tau and 1:55 4-repeat tau are all relatively close to the control (no-tau) point, the two 3-repeat tau conditions decrease the microtubule growth rate while the 1:55 4-repeat tau condition increases it as compared to the no-tau condition. Additionally, any increase in the tau:tubulin ratio beyond these low levels causes a relatively large increase in growth rate, since the distance between the no-tau point and all other tau points is relatively large. Thus, there are two clusters of growth rates rather than a simple linear relationship, consistent with a threshold effect. Further, as the tau:tubulin ratio increases (for both tau isoform classes), the difference with the no-tau point increases. Finally, for any given tau:tubulin ratio, 4-repeat tau is always more distant from the no-tau point than 3-repeat tau is; this demonstrates that 4-repeat tau is a more potent regulator of microtubule dynamics than 3-repeat tau. Thus, these data establish the validity of our automated life history analytical method and the two-dimensional embedding method.Figure 3Growth rate (sample 1). Embedding of the growth rates of tau conditions for sample 1 (corresponding to Table 1). The distortion is 1, indicating no error in embedding. The automatically computed growth rates maintain the relationship of the conditions as described in the Results section and in [19]. Distortion = 1.00.Figure 4 shows the plot for another set of samples corresponding to the values in Table 2. This second sample corresponds to tubulin preparation 1 mentioned in Table II of [19]. The low ratios of 3-repeat and 4-repeat tau:tubulin behave similar to the control (no-tau) point. The higher ratios cluster separately.Figure 4Growth rate (sample 2). Embedding of the growth rates of tau conditions for sample 2 (corresponding to Table 2). The distortion is 1, indicating no error in embedding. The automatically computed growth rates maintain the relationship of the conditions as described in the Results section and in [19]. Distortion = 1.00.Microtubule Growth Rate Distribution HistogramNext, we used two-dimensional embedding to compare the effects of 3-repeat tau and 4-repeat tau upon the distribution of growth rates within the growing population of microtubules. As demonstrated in [19], a histogram analysis of control populations of growing microtubules yields two pools – a more abundant and slower growing pool and a less abundant and faster growing pool. Based on fitting mixture of two Gaussians to the histograms, the authors concluded that both tau isoforms cause an increase in the abundance of the faster growing pool and a decrease in the abundance of the slower growing pool, with 4-repeat causing the population change at lower tau:tubulin ratios than 3-repeat tau. Other than these conclusions, there was no other comparison possible between the histograms.We subjected the growth rate distribution data to our two-dimensional embedding analysis method (Figure 5). Each distribution histogram had 19 bins (similar to the analysis in [19]), and the dissimilarities among the histograms were computed by the match distance [29]. Conceptually, the match distance takes into account both the height of a histogram bin and the spatial position of the bin in the histogram; two histograms that differ in far-off bins are more distant than histograms that differ in adjacent bins. The details of the procedure are presented in the Methods section. As was true for the growth rates (Figure 3), the histogram distribution data reveals that there are only minor differences among the control (no-tau) sample and low levels of tau (3-repeat tau at both 1:55 and 1:45 tau:tubulin ratio and 4-repeat tau at a 1:55 tau:tubulin ratio). Moreover, in a manner parallel to the growth rates, 3-repeat and 4-repeat tau regulate microtubule dynamics in different directions, as indicated by the fact that the 4-repeat tau (1:55 ratio) is closer to the no-tau point than it is to either of the 3-repeat tau (1:55 or 1:45) samples.Figure 5Growth rate distribution histogram (sample 1). Embedding of the microtubule growth rate distributions at varying tau:tubulin molar ratios for sample 1. The growth behavior of microtubules for low molar ratios of tau:tubulin for both 4-repeat and 3-repeat taus are similar to those in no-tau conditions. In higher molar ratios, however, the behavior is quite different. Distortion = 1.84.Additionally, it is also clear that 4-repeat tau is more potent than 3-repeat tau at any given tau:tubulin ratio (i.e., the distance between the 4-repeat tau point and the no-tau point is greater than the distance between the 3-repeat tau point and the no-tau point for all molar ratios). Finally, similar to the growth rate analysis in Figure 3, there are two clusters of behaviors rather than a continuum. One cluster contains the no-tau point and the lower tau:tubulin ratio samples and the other cluster contains the higher tau:tubulin ratio samples. Such non-linearity coupled with different functional effects could have significant mechanistic effects in the alternative RNA splicing class of tau FTDP-17 mutations in which relatively subtle increases in the 4-repeat tau concentration have dramatic consequences. By assessing the histogram landscape of the conditions, the two-dimensional embedding procedure complements the previous analyses using Gaussian mixture models [19]. The two-dimensional embedding plot is more sensitive in picking out the differences between a pair of conditions or among multiple conditions; on the other hand, it shows distances that lack physical meaning.Figure 6 shows the corresponding embedding plot of the growth rate distribution histograms for another set of samples. This second sample corresponds to tubulin preparation 1 mentioned in Table II of [19]. Similar to the case presented in Figure 5, the low ratios of 3-repeat and 4-repeat tau:tubulin cluster together with the control (no-tau) point. The higher ratios of tau:tubulin induce shifts in the growth rates.Figure 6Growth rate distribution histogram (sample 2). Embedding of the microtubule growth rate distributions at varying tau:tubulin molar ratios for sample 2. The growth behavior of microtubules for low molar ratios of tau:tubulin for both 4-repeat and 3-repeat taus are similar to those in no-tau conditions. In higher molar ratios, however, the behavior is quite different. Distortion = 1.69.Additional file 1 shows the effect of the number of bins on the embedding plots. Histograms were generated by varying the number of bins from 4 to 29 in variations of 5. The plots show only minor differences. In all of them, the lower tau:tubulin ratios (4-repeat tau at 1:55, and 3-repeat tau at 1:55 and 1:45) and the control (no-tau) point are far away from the higher tau:tubulin ratios.Microtubule Dynamics and Haar WaveletsFinally, we compared the two-dimensional embeddings of the Haar wavelet features [30] to 3-repeat tau, 4-repeat tau and the control (no-tau) samples. Wavelets [31] are powerful statistical tools that are used for a wide range of applications, including signal description and data compression. One of the main advantages of wavelets is that they offer a simultaneous localization in both time and frequency domains. Further, they can provide a multi-resolution view of the original time-series by changing the width of the \"window\" over which the coefficients are computed. Haar wavelets [30] are the simplest and the fastest to calculate among all the different types of wavelet functions. The specific window sizes and the details of how the dissimilarities among the conditions are computed are described in the Methods section. Additional file 2 shows the plots for the two different samples. The disparity in the two plots likely arises from the inherent variability in the biological data. The first plot (corresponding to the data set presented in Table 1) suggests two distinct clusters, one corresponding to the 3-repeat tau conditions and the other to the 4-repeat tau conditions, consistent with the notion that 3-repeat and 4-repeat tau might interact with microtubules in qualitatively distinct manners. The lack of similar behavior for the second data set (see Table 2) makes the conclusions from the plots tentative, requiring independent corroboration.We also used two-dimensional embedding to compare the effects of 3-repeat and 4-repeat tau with respect to the Markov Chain models. A Markov Chain (MC) [32] captures the underlying dynamics of the physical phenomena or entity by a generative model that emits a sequence of symbols. The primary advantage of Markov Chains over other models of time-series data is their ability to characterize an entire family of sequences. MCs are fairly easy to build, require a small set of sequences and allow very fast searching and comparison. There was no obvious clustering of points with respect to either the tau:tubulin ratio or 3-repeat tau versus 4-repeat tau (plot not shown). We used other time-series models as well, like the Lomb-Scargle periodograms [33,34] that can assess periodic behaviors (akin to Fourier analysis [35]) even in the presence of missing data and unequal sampling frequencies. Unfortunately, the embedding plot did not reveal any clear patterns, with the exception that the control (no-tau) point was on a distant corner of the plot and the tau samples with lower molar ratios of tau:tubulin are closer to the no-tau point than the samples with higher ratios (data not shown). Another class of models – the auto-regressive moving average (ARMA) models [36] – has often been used in analyzing time-series data. These models assume that the data is stationary, i.e., both the mean and the variance is fixed. Since the microtubules are clearly growing, we did not consider these models.DiscussionThis work addresses the need of the biological research community for rigorously quantitative and generally applicable computational tools to compare the complex behaviors of individual members of groups of molecules, cells or even organisms. Presently, the vast majority of such comparisons are performed manually, or \"by eye\". As such, they are time-consuming, susceptible to inadvertent bias and errors and can be insensitive to subtleties. Using the regulatory effects of the microtubule associated protein tau upon the dynamic behavior of microtubules as a system of study, we have developed a novel modeling and visualization strategy allowing investigators (i) to assess the relative degree of similarity/dissimilarity among individual tau isoforms with respect to numerous parameters of interest under varying experimental conditions, and (ii) to visualize all the conditions with respect to each other. More importantly, the same computational strategy should be generally applicable to a great many other applications.The validity of the two-dimensional embedding strategy presented in this paper is established by comparing the plot presented in Figure 3 with the growth rate data in Table 1. The relative positions of all points in Figure 3 are in complete agreement with the quantitative growth rate data determined both automatically and manually. Additionally, the semi-quantitative analysis of the histograms shown in Figure 4 of [19] are confirmed and extended by the more rigorous quantitative analysis leading to the two-dimensional embedding plot shown in Figure 5. In this case, 19 different bins of microtubule growth rates were integrated into the analysis for each of the nine experimental conditions tested. The resulting two-dimensional plot in Figure 5 presents the investigator with novel perspectives on the data set, including the existence of two clusters of histogram distributions based on growth rate as well as the distinct behavior of low ratios of 3-repeat tau:tubulin relative to all other tested reactions.Finally, although the molecular mechanisms underlying behaviors suggested by various statistical models may not be clear, these models could suggest mechanisms that could not be drawn using the standard manual analytical methods generally utilized in biological investigations. Indeed, one of the most important and generally applicable features of our computational strategy is the ability to detect subtle relationships between different molecules or conditions that might escape manual investigation.ConclusionIn this manuscript, we present (i) an automated method for quantitatively characterizing microtubule dynamics as a function of time, and (ii) a novel and generally applicable computational tool for two-dimensional visualization and modeling of entities of interest for comparative studies. Comparison of our automated tracking method with manually acquired data demonstrates its accuracy. This tool greatly increases the rate at which microtubule tracking data can be acquired as well as improve upon its objectivity and accuracy. Our embedding strategy accurately recapitulates and extends previous biological observations that were collected and analyzed manually. Importantly, our methods facilitate the integration of sophisticated statistical modeling with biological investigations, which should promote novel and deeper mechanistic insights into biological phenomena as well as the development of testable hypotheses for subsequent investigation. In the future, we anticipate applying these methods to compare wild-type tau action versus various tau mutants causing neurodegeneration and dementia, seeking to identify novel mechanistic effects. Additionally, we envision using new models and embedding strategies.MethodsModelingThe different models described in this section capture different characteristics of microtubule dynamicity. Comparison of conditions across these models highlights different features of tau action.Microtubule EventsThree kinds of events are used to characterize microtubule dynamics: growth, shortening and attenuation (\"pause\"). Each kind of event can be simple or complex. An event is simple when it is characterized between two consecutive tracked time-points. Simple events are coalesced together to form bigger complex events. Complex events, therefore, can be defined over a contiguous set of more than two time-points. Identification of simple events are easy, but identifying the start and end points of complex events require sub-sequence analysis.The simple events are classified in the following manner. The different parameters for defining these events used in this particular study are indicated next. An event is a simple growth if and only if: (i) the rate of increase of microtubule length is at least 0.5 μm/min, and (ii) the increase in length is at least 0.05 μm. The corresponding parameters for a simple shortening event are: (i) the rate of decrease of length is at least 0.5 μm/min, and (ii) the decrease in length is at least 0.5 μm. A simple attenuation event must have (i) a rate of change of length outside the range for simple growth and simple shortening events, i.e., between -0.5 μm/min and +0.5 μm/min, and (ii) a total time duration of at least 4 s. Any event that does not fall in any of the above categories are excluded from the analysis. Due to errors in human tracking and image resolution issues, such events are likely to be part of the input noise, and are hence, discarded. Simple events are used for Markov Chain analysis.The complex events have their corresponding parameter cut-offs as well. However, the more important consideration in the analysis of complex events is the identification of where it starts and where it ends. A survey of such methods from the time-series literature can be found in [37]. These methods have been successfully used to segment time-series streams into different partitions in various application domains, most notably for stock market analysis. An interesting way to combine different segmentation outputs has been proposed in [38]. However, none of these methods have employed priority rules to analyze adjacency relationships.We now describe our bottom-up approach of identifying complex events by merging together simple events. First, all consecutive simple events of the same type are merged together to form a longer complex event of the same type. Next, each complex event is subjected to the rule set for classifying into growth, shortening, and attenuation. An event that does not pass any of the three rule sets is classified temporarily as an error. Also, the cause of its failure is noted. More specifically, any event where there is an increase in length but which cannot be classified as a growth is assigned into two kinds of failure: (i) rate, where it did not pass the growth rate threshold, and (ii) length, where it did not pass the growth length threshold. The failed shortening events are classified similarly. Note that there are no attenuation failure classes.The priority rules are applied next. A growth rate failure event is most likely to be part of an attenuation. Thus, its neighbors are examined and if possible, it is combined with adjacent attenuation events to form a bigger attenuation event. If this fails, then attempts to incorporate with neighboring growth events, if any, are made. If, however, the growth failure event is due to the length cutoff and not the rate, then this event is most likely to be part of a growth event. The error in length may be due to human tracking and image resolution issues. Hence, attempts to combine this with neighboring growth events are first carried out. The rules for absorbing the shortening failure events are similar.The complex event cut-offs are: (i) Growth: rate ≥ 0.5 μm/min, length ≥ 0.06 μm; (ii) Shortening: rate ≤ -0.5 μm/min, length ≤ -0.6 μm; and (iii) Attenuation: rate between -0.5 μm/min and +0.5 μm/min, time ≥ 30 s. The growth rates and the growth distributions are calculated using the complex events.Figure 2 shows a comparison of the manually marked complex events and the automatically measured ones. The solid line indicates the simple events. As evident from the figure, these tracked lengths are noisy. The complex events get rid of the noise by smoothing over a range of simple events. However, while the automatic method marks three events – two growth events separated by a shortening event – a human may simply mark the entire time-history as a single growth event. Clearly, this human bias will differ from one experimenter to another, and may even vary from time to time. Note that this explains why growth rates obtained from the automatic measurements vary (become slightly higher) from those obtained through the manual method.The parameters for the different events have been chosen empirically by biologists based on experimenting with different kinds of microtubule samples. The event definitions have been used consistently and have become the de facto \"industry standard,\" as evident from [4].Growth RateThe growth rate for a particular experimental condition was calculated as the average of the growth rates of all the complex growth events of the microtubules for that condition.In order to understand whether the differences between the automatically computed growth rate values using the above event analysis technique and the manually measured ones are statistically significant, we calculated the p-values in the following way. Two groups were formed, one with the automatically identified growth events, and the other with the manually marked growth events. We then performed a t-test [39] to determine whether the means of the growth rates of the two groups are different. The p-values thus obtained are reported in Tables 1 and 2.Growth Rate Distribution HistogramFor each condition, a growth rate distribution histogram was computed in the following manner. The rates for the complex growth events were divided into 18 bins of width 0.4 μm/min each (consistent with analysis by [19]), starting from 0.5 μm/min up to 7.7 μm/min. Once again, these parameters conform to the standards set in the microtubule event analysis literature [4]. All the higher growth rate events were collected in another bin. Thus, the histograms had 19 bins in total. The bin heights were normalized such that they add up to 1, yielding a growth rate distribution. In order to generate histograms with a fixed number of bins, say b, the width of each bin was specified as 7.7−0.5bμm/min.Haar WaveletsWavelets are mathematical functions that describe time-series data in terms of various frequency components with resolutions matched to their scales [31]. The orthonormal basis vectors, called the mother wavelets, that describe the various wavelet components are given by:ψs,l(x) = 2-s/2ψ(2-s x - l)where s denote the scaling factor and l the localization in time. The Haar wavelet basis functions [30] are the simplest:ψ(x)={+10≤x<1/2−11/2≤x<10otherwiseHaar wavelets are also the fastest to calculate with respect to other wavelet bases. They work by progressively retaining the most important parameters of a signal. The first coefficient is the \"sum\" (actually, scaled average) of the entire signal and the next gives the \"detail\" (difference of the two halves) of the signal. The later coefficients give more and more details about each half of the signal they model.In general, more wavelet parameters mean more detailed description; however, they also mean more data and more noise. Further, the error in embedding is directly proportional to the number of parameters of the original data. Thus, 16 coefficients of each microtubule time-series were retained. These 16 coefficients were then averaged over all the microtubules from a particular experimental condition to yield the coefficients for that condition.Markov ChainA Markov Chain (MC) [32] is a discrete time stochastic process that models the observations of a dynamic system (such as the growth or the shortening of a microtubule) as the states of the system. The number of states is finite and there is a state corresponding to each observation symbol. In a first-order MC, the probability of occurrence of the future state (or observation) depends only on the current state; past states are inconsequential. This property is called the Markov property. (In a kth order Markov Chain, the future state depends on the current state and k - 1 past states.)Formally, an MC λ is defined as:λ = {n, π, τ}where n is the number of states, π is the start state probability vector of length n, and τ is the n × n transition matrix; π(u) denotes the probability of being in state u in the first time-step; and, τ (u, v) denotes the probability of reaching state v from state u in a single time-step.In the work of [19], microtubules were in a non-equilibrium phase, exhibiting very little shortening and many microtubules never shortened at all. Therefore, the microtubule events were discretized into two symbols: G for growth, and N for non-growth (shortening or attenuation). The Markov Chains were built with these two states – growth and non-growth. Since shortening events were very rare, modeling it as a separate state would have lacked statistical validity.The transition probabilities for the MCs were estimated in the following manner. Every microtubule time-series was denoted as a string of symbols, with each symbol representing a simple event. Then, pairs of consecutive symbols (states) were read and appropriate entries in the transition matrix were incremented. When all the microtubules in an experimental condition were processed, the transition matrix was normalized such that the sum of transition probabilities from each state form a probability distribution (adds up to 1). The start state probabilities were estimated in a similar manner by reading the first symbol for every microtubule time-series; if it is growth, the entry for G is incremented, otherwise that for N is incremented. Finally, normalization was performed such that the probabilities add up to 1. Since most of the microtubules started with growth, these vectors were very close to [1, 0].Lomb-Scargle PeriodogramsThe periodicity analysis of the microtubule data was performed by extracting Lomb-Scargle coefficients [33,34] from each time-series. Lomb-Scargle periodograms capture the different frequency components in a time-series and can handle missing values and unequal sampling intervals. Four low frequency components (corresponding to periodicities of 4, 8, 16, and 32 s) were retained for each microtubule. The Lomb-Scargle coefficients for the condition were computed as the average of the corresponding coefficients of the individual microtubules.Dissimilarity FunctionsIn order to compare a pair of models, an appropriate similarity or dissimilarity function is necessary. The dissimilarity or distance measure is used to compute the distance matrix among the conditions; this distance matrix is then embedded in a two-dimensional vector space as described later in the Visualization section.Growth RateThe dissimilarity between a pair of conditions with respect to the growth rates was measured by their difference. The difference can be also viewed as a Minkowski form of distance or Lk norm. The Lk norm between two vectors p and q of length k each is defined as:Lk(p,q)=[∑∀i|pi−qi|k]1/kGrowth Rate Distribution HistogramGrowth rate histograms can be viewed as vectors and Lk norms can be employed to capture the dissimilarity between a pair of histograms. These measures, however, do not capture the relationship among the different histogram bins. For example, suppose there are three bins in each histogram corresponding to low rate of growth, medium rate of growth and high rate of growth. If A = [1, 0, 0], B = [0, 1, 0] and C = [0, 0, 1], then Lk norms treat these histograms as equidistant from each other, even though A should be more different from C than B. To capture such spatial properties of the bins, match distance [29,40] was employed.To calculate the match distance between a pair of histograms p and q, a distance matrix among the bins of the histogram are specified – the distance between two bins i and j is cij = |i - j|. The match distance is defined as the minimum work required to be done in order to transform the histogram p into the histogram q by moving values or \"flows\" from the bins of p to those in q and vice versa. Having a flow fij from bin i of p to bin j of q or vice versa is considered as cij·fij amount of work. Finding the match distance then reduces to finding the flows fij such that the total work done is minimum. The minimum work done is the match distance:MD(p,q)=min⁡f{∑i,jcijfij}For the example histograms A, B, and C mentioned above, the match distances are MD(A, B) = 1, MD(B, C) = 1, and MD(A, C) = 2. Clearly, this captures the relatively larger dissimilarity of A from C as compared to that from B.For one-dimensional histograms where the sum of the bin values add up to the same number (here, 1), match distance can be calculated more easily as the L1 distance between the cumulative bin values of the two histograms:MD(p, q) = L1(P, Q)where Pi=∑j=1ipi and Qi=∑j=1iqi are the cumulative histogram bin values.Haar WaveletsSince the relative importance of the wavelet parameters differ, a simple distance function such as L1 would be inappropriate. Coefficients that summarize the entire time-series, such as the \"sum\" value and the overall \"detail\" value is more important, and therefore, should get higher weights than the coefficients describing parts of the time-series.Thus, in order to determine the dissimilarity between two conditions with respect to their Haar wavelet coefficients, we used the weighted L1 norm or the weighted Manhattan distance. The levels of the wavelet tree were weighted such that the overall sum and the overall detail coefficient were the most significant values, the next level detail coefficients getting an exponentially lower weight and so on. The weight vector, of length 16, was [8, 8, 4, 4, 2, 2, 2 2, 1, 1, 1, 1, 1, 1, 1, 1]. For two vectors p and q, and a weight vector w, all of length k, the weighted L1 distance between p and q is measured as:L1(p,q)=∑i=1kwi|pi−qi|The L2 norm or the Euclidean distance was applied to measure the distances between a pair of conditions for both the Markov Chain parameters and the Lomb-Scargle coefficients.VisualizationThe distances among the experimental conditions, calculated by using the above methods, were visualized by plotting the conditions onto a two-dimensional vector space. This allows for easy comparison of the conditions and immediate comprehension of the structure of the data. The aim of the embedding method is to assign coordinates such that the Euclidean distance between any pair of conditions in the embedded space is as close as possible to the dissimilarity calculated between their models. The method can embed a given set of points into any dimensional space; here, we have chosen two for easy visualization purposes.Formally, suppose there are two models, α and β, and the dissimilarity between them is d(α, β) according to some dissimilarity function d. If the embeddings of these two models in the two-dimensional vector space (x, y space) is given by e(α) = (xα, yα) and e(β) = (xβ, yβ), then the aim of the embedding function is to choose the coordinates e(α) and e(β) such that the relative difference between L2(e(α), e(β)) and d(α, β) is minimum. When there are n such models, the embedding function should be chosen such that the relative cumulative difference for all the n(n - 1)/2 pairs is minimized.Principal component analysis (PCA) [28] can also be used to project data onto a two-dimensional space. PCA chooses the axes along which the original data shows the highest variance. It does not take into account the distances among the points. More importantly, PCA cannot work with any general distance matrix and is used mostly as a dimensionality reduction technique.We used the Sammon projection method [27] as the embedding procedure. This method has been successfully used to embed proteins on a two-dimensional space for clustering purposes [41]. The method starts with a random point (random x, y coordinates) for each model. In each iteration, the points are updated according to a steepest descent algorithm such that the following error function E(x, y), which measures the relative differences between the original distances and the embedded distances, is decreased.E(x,y)=1c∑∀α,β,α≠β[(d(α,β)−L2(e(α),e(β)))2d(α,β)]wherec=∑∀α,βd(α,β)In each iteration, a correction step is added to every dimension of every point. The direction of the correction is towards the gradient of the error. The coordinates of the point α in iteration i + 1 are updated as follows:xα(i+1)=xα(i)−f×∂E(i)/∂x|∂2E(i)/∂x2|yα(i+1)=yα(i)−f×∂E(i)/∂y|∂2E(i)/∂y2|where E(i) is the error after iteration i and f is a factor to control the step sizes. We used f = 0.2. The method stops after a certain number of steps or when there is no significant improvement in the error. We stopped the iterations either when the change in error went below 0.01% or up to a maximum of 1000 steps. The number of steps was set as an additional check in order to come out of any local error problems, e.g., oscillating error values. In practice, after 250–300 iterations, the error stopped changing, and the algorithms stopped. In addition, in order to counter the problem of bad initialization, the algorithms were run 5 times for each embedding and the one with the lowest error was picked. The final coordinates or the directions of the axes do not have any significance; only the Euclidean distances among the embedded points matter.For any dimensionality reduction or embedding technique, an important measure of quality is distortion. Distortion measures the largest amount of discrepancy from an original distance value to the corresponding embedded distance. It is measured asdistortion=max⁡{original distembedded dist}min⁡{original distembedded dist}where original dist refers to an original dissimilarity measure between two models and embedded dist refers to the Euclidean distance between the corresponding embedded points. For ideal embeddings, where all the original distances have been maintained exactly, the distortion is 1. For others, the distortion is greater than 1. In general, lower the distortion value, better the embedding. The individual distortions are measured by the ratio of embedded dist to original dist.For models with only a single parameter, any dissimilarity between a pair of them is equal to the difference between their single parameters. Such distance matrices can be always embedded into two dimensions with distortion equal to 1. The models have to be simply embedded as points on a straight line with the order and the distances maintained, e.g., as (parameter, 0) or (parameter/2, parameter/2) points. In our implementation, we have not forced this explicitly; the method itself converges to a straight line plot. For models with two parameters, if the dissimilarity function is Euclidean, then, it is again possible to devise an embedding with distortion 1. The original parameter values will form the coordinates in the embedded space. For higher number of parameters or with other dissimilarity functions, in general, it is not possible to design embeddings with distortion 1. The distortions of each of the graphs are mentioned in the captions. Additional file 3 reports the individual distortions for each of the distances for all the embeddings.Authors' contributionsAB built the models, computed the pairwise distances, and embedded them on two dimensions for comparisons. AL, SL and MG collected the data and manually tracked the microtubules. AKS advised on the choice of the models and the distance functions. SCF and LW provided the biological interpretations. AB and SCF wrote the manuscript with inputs from the other authors. All the authors read and approved the final manuscript.Supplementary MaterialAdditional file 1This file shows the effect of number of bins on the growth rate distribution histograms.Click here for fileAdditional file 2This file shows the two-dimensional embedding plots of the Haar wavelet coefficients of microtubules with varying tau:tubulin molar ratios for both the samples.Click here for fileAdditional file 3This file shows the individual distortions of all the pairwise distances between the conditions for all the different models described.Click here for file\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2533034.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533034",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533034\nAUTHORS: Mong-Ping Shyong, Fenq-Lih Lee, Ping-Chang Kuo, Ai-Ching Wu, Huey-Chung Cheng, Show-Li Chen, Tao-Hsin Tung, Yeou-Ping Tsao\n\nABSTRACT:\nPurposeTo evaluate the efficacy of recombinant adeno-associated virus (rAAV) vector expressing mouse angiostatin (Kringle domains 1 to 4) in reducing retinal vascular leakage in an experimental diabetic rat model.MethodsrAAV-angiostatin was delivered by intravitreal injection to the right eyes of Sprague-Dawley rats. As a control, the contralateral eye received an intravitreal injection of rAAV-lacZ. Gene delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Diabetes was induced by intravenous injection of streptozotocin (STZ). Vascular permeability changes were evaluated by extravascular albumin accumulation and leakage of intravenous-injected fluorescein isothiocynate-bovine serum albumin (FITC-BSA). Effects of rAAV-angiostatin on expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), occludin, and phospho-p42/p44 MAP kinase in retina tissue were analyzed by western blotting.ResultsThe rAAV-angiostatin injections led to sustained angiostatin gene expression in retina as confirmed by RT-PCR, and reduced extravascular albumin accumulation in STZ-induced diabetic retina. Further, rAAV-angiostatin significantly decreased intravascularly injected FITC-BSA leakage at 5 days (p=0.001), 10 days (p<0.001), and 15 days (p=0.001) after STZ-induced diabetes, as compared to the control eyes receiving rAAV-lacZ. Expression of VEGF and phosphorylation of p42/p44 MAP kinase in retina was reduced by rAAV-angiostatin at day 1 (p=0.043 for both VEGF and phospho-p42/p44 MAP kinase) after STZ-induced diabetes compared with rAAV-lacZ eyes. rAAV-angiostatin reduced retinal occludin loss at 10 days after STZ-induced diabetes (n=5, p=0.041). There was no significant difference in retinal PEDF expression between eyes injected with rAAV-angiostatin and rAAV-lacZ.ConclusionsIntravitreal delivery of rAAV-angiostatin reduces vascular leakage in an STZ-induced diabetic model. This effect is associated with a reduction in the retinal occludin loss induced by diabetes and downregulation of retinal VEGF and phosphor-p42/p44 MAP kinase expression. This gene transfer approach may reduce diabetic macular edema, providing protection in diabetic patients at risk for macular edema.\n\nBODY:\nIntroductionDiabetes mellitus is the most prevalent endocrine disease in developed countries [1], and diabetic retinopathy is the leading cause of blindness in the world [2,3]. Blood-retinal barrier (BRB) breakdown, increased vascular permeability and vascular leakage are early complications of diabetes and a major cause of diabetic macular edema [4-6]. As there is no satisfactory or noninvasive therapy, diabetic macular edema is a major cause of vision loss in diabetic patients [7].An ideal treatment strategy would be to deliver a therapeutic gene with a vector that could confer long-term transgene expression and tissue protection with a single administration. We have previously reported that a recombinant adeno-associated virus vector expressing angiostatin (rAAV-angiostatin) suppressed laser-induced choroidal neovascularization [8]. Recently, an effect of angiostatin in reducing vascular permeability in the retina in diabetic and oxygen-induced retinopathy models was reported [9].Angiostatin (Kringles 1 through 4) is a proteolytic fragment of plasminogen [10]. It was identified as a potent angiogenic inhibitor, which blocks neovascularization and suppresses tumor growth and metastases [10,11]. Angiostatin specifically inhibits proliferation, induces apoptosis in vascular endothelial cells [12], and downregulates vascular endothelial growth factor (VEGF), the latter via inactivation of the p42/p44 MAP kinase pathway [9,13,14]. We also noted that some proteolytic fragments of plasminogen can induce upregulation of pigment epithelium-derived factor (PEDF) expression, a potent angiogenic inhibitor in experimental diabetes [15].BRB breakdown may be due to disassembly of unique proteins that constitute the functional vascular endothelial tight junction [16-18]. VEGF is a potent angiogenic factor [13], whose overproduction in the retina has been noted in the development of vascular hyperpermeability in diabetes [14]. Furthermore, VEGF affects the tight junction protein occludin, inducing occludin phosphorylation [19] and redistribution [20]; resultant occludin reduction is associated with BRB breakdown in diabetes [21].The present study was designed to examine the transgenic expression of rAAV-angiostatin in the eye and its effect on vascular permeability in the streptozotocin (STZ)-induced diabetic model. Since it has been demonstrated angiostatin can induce the downregulation of VEGF through the blockade of phosphorylation of p42/p44 MAP kinase [9,13,14], we also studied the relationship between rAAV-angiostatin, p42/p44 MAP kinase, occludin, VEGF, and PEDF in this model.MethodsAnimalsMale Sprague-Dawley (SD) rats (Charles River Laboratories, WilmingtonMA) weighing approximately 200 g on arrival were used in this study. The animals were cared for in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experimental procedures used aseptic sterile techniques and were approved by the Animal Care and Use Committee of the Mackay Memorial Hospital.Generation of rAAV-angiostatincDNA coding for angiostatin was amplified by polymerase chain reaction (PCR) according to a published report [22]. rAAV encoding mouse angiostatin cDNA or lacZ were constructed by using a three-plasmid cotransfection system as described previously [22-24]. Titers of rAAV-angiostatin and rAAV-lacZ were determined by dot blot hybridization using angiostatin cDNA and lacZ as probes [25].Intravitreal injections of rAAV-angiostatinAfter being anesthetized, each animal received an intravitreal injection of rAAV- angiostatin (5 μl, 1.5x1010 viral particles) as described previously [26]. The contralateral eye of each rat was injected with rAAV-lacZ to serve as a control.Experimental DiabetesExperimental diabetes was induced three weeks after intravitreal injection of rAAV. Diabetes was induced with a single 60 mg/kg intravenous injection of streptozotocin (Sigma-Aldrich, St. Louis, MO) in 10 mM citrate buffer, pH 4.5. Animals that served as nondiabetic controls received an equivalent amount of citrate buffer alone [21] Twenty-four h later, rats with blood glucose levels higher than 250 mg/dl were deemed diabetic. These diabetic rats received 6-8U NPH insulin (Lilly, Indianapolis, IN) once a week to prevent ketoacidosis. Just before experimentation, blood glucose levels were measured again to confirm diabetic status.Reverse transcription-polymerase chain reactionExpression of rAAV-angiostatin in retina was confirmed by RT-PCR according to a protocol described in reference [8]. Each rat eye that was previously injected with rAAV-angiostatin and rAAV-lacZ was enucleated, and chorioretinal tissues were harvested for RT-PCR at 1, 5, 10, and 15 days after induction of experimental diabetes. The cDNA was synthesized using oligo(dT) primer and 200 IU transcriptase (SuperScript II; Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. PCR amplification was performed with two oligonucleotide primers, 5'-CAG CAA TGC GTG ATC ATG-3' and 5'-TGG AGA TTT TGC CCT CAT AC-3'. As a control, PCR amplification was performed for glyceradehyde-3-phosphate dehydrogenase (GAPDH) with two oligonucleotide primers, 5'-GGA AGG GCT CAT GAC CAC AG-3' and 5'-CCT TTA GTG GGC CCT CGG-3'. To rule out the possibility that gene amplification products were derived from amplification of contaminating angiostatin genomic DNA, we treated the total RNA with RNase free DNase I (Qiagen, Valencia, CA) before RT-PCR.Immunofluorescence assayFive-μm-thick retinal tissue sections from formalin-fixed, paraffin-embedded blocks were transferred to positively charged slides to be used for staining. Sections were dewaxed in xylene and progressively hydrated [27]. They were then washed three times with PBS and a 1:400 dilution of polyclonal rabbit antihuman albumin antibody (DAKO Diagnostics. Mississauga, ON, Canada) was applied. A fluorescein isothiocyanate (FITC)-conjugated antimouse IgG was used as a secondary antibody. The results were viewed with a fluorescence microscope (Zeiss Axioplan HBO100, Oberkochen, Germany).Measurement of leakage with intravascular injected FITC-BSARetinal vascular leakage was measured using the intravascular injected FITC-BSA as previously described [21,28] with some modifications. After induction of anesthesia, the rats received tail vein injections of 100 mg/kg FITC-bovine serum albumin (FITC-BSA, Sigma-Aldrich). The animals were sacrificed 20 min later, and their eyes were removed, embedded in OCT medium, and snap-frozen in liquid nitrogen. The plasma was collected and assayed for fluorescence with an SPEX fluorescence spectrophotometer (Molecular Devices, Sunnyvale, CA) based on standard curves of FITC-BSA in normal rat plasma. Frozen retinal sections (6 μm thick) collected every 60 μm were viewed with a Zeiss Axioplan HBO100 fluorescence microscope. Images from six retinal nonvascular areas (200 μm2) in each section were collected. Quantification of FITC-BSA fluorescence intensity was calculated by computer software Q-win (Leica, Wetzlar, Germany) and normalized to plasma fluorescence intensity for each animal.Western blot analysis of VEGF, PEDF, occludin, and p42/p44 MAP kinaseRats were sacrificed, and their eyes were removed for western blotting analysis at 1, 5, 10, and 15 days after STZ treatment. The chorioretinal tissue was harvested, and the soluble fractions were prepared by homogenizing the retina in Eppendorf tubes containing RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF, 1 mM EGTA, 1 mM PMSF, 1 mg/ml leupeptin, and 1 mg/ml pepstatin A). Proteins (50 μg) were extracted for electrophoresis on 10% SDS-polyacrylamide gels. The membranes were incubated with antibody specific to VEGF [29,30], PEDF [31], p42/p44 MAP kinase [21] (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and occludin [20] (Zymed, San Francisco, CA). The results were semiquantified by densitometry (Fujifilm LAS3000, Tokyo, Japan) and normalized to actin levels.Cell culture and rAAV-angiostatin infectionTo further evaluate whether angiostatin could induce the expression of PEDF in endothelial cells, we infected human umbilical vein endothelial cells (HUVEC-2C, Cascade Biologics, Portland, OR) with rAAV-angiostatin. The HUVEC-2C cells were cultured in medium 200 (Cascade Biologics) containing low serum growth supplement. All cells were supplemented with 1% penicillin-streptomycin and maintained at 37 °C and 5% CO2. Confluent cells obtained during the fifth passage were used for rAAV-angiostatin infection. The HUVEC-2C cells were infected by rAAV-angiostatin in Dulbeco's modified Eagle's medium for two days. Cell lysates were then prepared and analyzed for PEDF by western blotting as described in the previous paragraph.Statistical AnalysesThe results are expressed as the mean±SD. Retinal FITC-BSA fluorescence intensity was analyzed in serial retinal sections from four rats at 5, 10, and 15 days after induction of diabetes. Due to skewed distributions, data were subjected to logarithmic transformation for analysis. Differences of retinal FITC-BSA fluorescence intensity between eyes receiving rAAV-angiostatin and rAAV-lacZ injection at 5, 10, and 15 days after induction of diabetes were analyzed by a paired-sample Student's t test. The retinal expression of VEGF, PEDF, phosporylation of p42/p44 MAPK, and occludin were analyzed by the Wilcoxon signed-rank test. All p-values are two-tailed, and differences were considered to be statistically significant for p<0.05.ResultsGene delivery by rAAV-angiostatinThere was no angiostatin gene expression in normal control eyes (Figure 1, lane A) and eyes injected with rAAV-lacZ at 1, 5, 10, and 15 days after induction of diabetes (Figure 1, lanes B-E). In the eyes injected with rAAV-angiostatin, angiostatin gene expression was detected at 1, 5, 10, and 15 days after induction of diabetes (Figure 1, lanes F-I). As an internal control, expression of GAPDH was detected in normal control eye and eyes receiving both rAAV-angiostatin and rAAV-lacZ injections (Figure 1, lanes A-I).Figure 1RT-PCR analysis of angiostatin cDNA in chorioretinal tissue. The eyes previously injected with rAAV-lacZ (lanes B to E) and rAAV-angiostatin (lanes F to I) were enucleated and chorioretinal tissues were harvested for RT-PCR at 1, 5, 10, and 15 days after induction of experimental diabetes. Lane A is the control eye. Lanes B and F are 1 day after diabetes induction. Lanes C and G are 5 days after diabetes induction. Lanes D and H are 10 days after diabetes induction. Lanes E and I are 15 days after diabetes induction. There was no angiostatin gene expression in the control eye (lane A) and eyes injected with rAAV-lacZ (lanes B to E). In the eyes injected with rAAV-angiostatin, angiostatin gene expression was detected (lanes F to I). As an internal control, expression of GAPDH was detected in normal control eye and eyes receiving both rAAV-angiostatin and rAAV-lacZ injections (lanes A to I). \"M\" indicates molecular weight markers.Induction of experimental diabetesAnimals (n=12) with a blood glucose exceeding 250 mg/dl were selected for inclusion in the diabetic group. All diabetic animals had higher blood glucose levels and reduced body weight gain at 5, 10, and 15 days after induction of diabetes compared to age-matched, nondiabetic control animals (Table 1).Table 1Animal physiological variables.After streptozotocin induction of diabetesBaseline (n=4)5 days (n=410 Days (n=4)15 Days (n=4)sampleBW (g)Blood sugar (mg/dl)BW (g)Increase in body weight (%)Blood sugar (mg/dl)BW (g)Increase in body weight (%)Blood sugar (mg/dl)BW (g)Increase in body weight (%)Blood sugar (mg/dl)Age-matched control rat248+14139+13260+134.8141+11294+1018.5154+12312+925.8143+11STZ-induced diabetic rat254+11143+9257+102.4352+15270+147.7377+13280+1111.6396+15Animals were made diabetic by a single streptozocin (STZ) injection (65 mg/kg) in 1 mmol/l sodium citrate buffer, pH 4.5. All diabetic animals had higher blood glucose levels and reduced body weight gain compared to age matched, non-diabetic control animals.rAAV-angiostatin influence on vascular permeabilityOne week after induction of diabetes, the immunofluorescence assay, using anti-albumin antibody, disclosed staining only inside blood vessels in normal control eyes (Figure 2A). Increased extravascular albumin in the retinal parenchyma was seen in the eyes of diabetic animals receiving rAAV-lacZ (Figure 2B). In the diabetic animals receiving rAAV- angiostatin, however, albumin staining was observed only inside blood vessels (Figure 2C).Figure 2Representative retinal sections following immunostaining for albumin. Increased immunostaining was present throughout the retina one week after STZ-induction of diabetes in eyes receiving rAAV-lacZ injection (B) compared to the normal Sprague-Dawley rat (A) where staining was restircted to blood vessels. C: Intravitreal injection of rAAV-angiostatin decreased immunostaining in the retina one week after induction of diabetes. Magnification X200. IPL denotes the inner plexiform layer, INL indicates the inner nuclear layer, and ONL marks the outer nuclear layer.To confirm the effect of rAAV-angiostatin on vascular permeability, we examined animals injected with intravenous FITC-BSA 5, 10, and 15 days after induction of diabetes. Figure 3 shows representative micrographs of the eyes of normal control and STZ-induced diabetic rats. FITC-BSA fluorescence is limited to the vasculature in the normal retina (Figure 3B) and diffusely increased throughout the retinal parenchyma at 5 days after STZ-induced diabetes in eyes receiving rAAV-lacZ injection (Figure 3C). Increased fluorescence intensity throughout the retinal parenchyma is still present at 10 (Figure 3D) and 15 (Figure 3E) days after STZ-induced diabetes in eyes receiving rAAV-lacZ injection. Little fluorescence was present in the retinal parenchyma in eyes receiving rAAV-angiostatin injection (Figure 3F) at 5 days after induction of diabetes. Retinal parenchyma fluorescence at 10 (Figure 3G) and 15 (Figure 3H) days after induction of diabetes in eyes with rAAV-angiostatin injection was decreased as compared with eyes that received the rAAV-lacZ injection.Figure 3FITC-BSA fluorescence in normal and streptozotocin (STZ)-induced diabetic rat retina. A: Hematoxylin and eosin staining of control retina. B: FITC-BSA fluorescence is limited to the vasculature in the normal retina and in C is diffusely increased throughout the retinal parenchyma at 5 days after STZ-induced diabetes in eyes receiving rAAV-lacZ injection. Increased fluorescence intensity throughout the retinal parenchyma is still present at 10 (D) and 15 (E) days after STZ-induced diabetes in eyes receiving rAAV-lacZ injection. F: Little fluorescence was present in the retinal parenchyma in eyes receiving rAAV-angiostatin injection at 5 days after induction of diabetes. Retinal parenchyma fluorescence at 10 (G) and 15 (H) days after induction of diabetes in eyes with rAAV-angiostatin injection was decreased as compared with eyes with rAAV-lacZ injection. Original magnification was 200X.The retinal FITC-BSA fluorescence intensity was calculated and normalized to plasma fluorescence intensity by image analysis of serial sections (Figure 4). Four SD rats were represented by the number of sections to be examined at each timepoint. The mean retinal FITC-BSA fluorescence intensity in eyes receiving rAAV-angiostatin injection was 2.99±0.62 pixels at 5 days, 3.42±0.38 pixels at 10 days, and 3.30±0.40 pixels at 15 days after induction of diabetes. The retinal FITC-BSA fluorescence intensity in eyes receiving rAAV-lacZ injection was 3.59±0.31 pixels at 5 days, 3.77±0.51 pixels at 10 days, and 3.71±0.47 pixels at 15 days after induction of diabetes. Quantitative analysis showed that the fluorescence was decreased in eyes receiving rAAV-angiostatin as compared to eyes receiving rAAV-lacZ at 5 days (t=3.67, n=49, p=0.001), 10 days (t=3.94, n=51, p<0.001), and 15 days (t=3.52, n=56, p=0.001) after induction of diabetes.Figure 4Quantification of vascular leakage in experimental diabetes. FITC-BSA fluorescence intensity was measured by image analysis in serial retinal sections. Rats each received an intravenous injection of FITC-BSA were sacrificed at 5, 10, and 15 days after induction of diabetes. The average retinal FITC-BSA fluorescence intensity was calculated and normalized to plasma fluorescence intensity. The retinal FITC-BSA fluorescence intensity in eyes receiving rAAV-angiostatin injection was 2.99±0.62 pixels at 5 days, 3.42±0.38 pixels at 10 days and 3.30±0.40 pixels at 15 days after induction of diabetes. The retinal FITC-BSA fluorescence intensity in eyes receiving rAAV-lacZ injection was 3.59±0.31 pixels at 5 days, 3.77±0.51 pixels at 10 days and 3.71±0.47 pixels at 15 days after induction of diabetes. The normalized FITC-BSA fluorescence intensity in eyes receiving rAAV-angiostatin was decreased as compared to eyes receiving rAAV-lacZ at 5 days (t=3.67, n=49, p=0.001), 10 days (t=3.94, n=51, p<0.001), and 15 days (t=3.52, n=56, p=0.001) after STZ-induction of diabetes. The asterisk indicates a p less than or equal to 0.001. Four SD rats were represented by the number of sections (n) to be examined.rAAV-angiostatin influence on occludin lossRats receiving rAAV-angiostatin in their right eyes and rAAV-lacZ in their left eyes were sacrificed, and their eyes were enucleated for western blotting analysis at 1, 5, 10, and 15 days after induction of diabetes. The retinal occludin content in normal control eye was 42.35±2.67 pixels. No differences in retinal occludin content were detected between eyes receiving rAAV-lacZ and rAAV-angiostatin injection at 1 (rAAV-lacZ: 45.82±3.08 pixels, rAAV-angiostatin: 43.62±2.78) and 5 days (rAAV-lacZ: 38.96±2.22 pixels, rAAV-angiostatin: 40.65±3.46 pixels) after induction of diabetes. Ten days after diabetes induction, retinal occludin content in eyes that received rAAV-lacZ was 11.35±3.57 pixels and 34.73±3.17 pixels in eyes that received rAAV-angiostatin occludin, a statistically significant reduction (n=5, p=0.041, Figure 5). The retinal occludin content in rAAV-lacZ-treted eyes was higher (26.32±3.46 pixels) at 15 days after induction of diabetes than it was at 10 days, and there was no difference in rAAV-angiostatin-treated animals (29.21±2.94 pixels).Figure 5The effect of rAAV-angiostatin gene transfer on retinal occludin expression 10 days after STZ-induction of diabetes The rats received intravitreal injection of rAAV-angiostatin in the right eyes and rAAV-lacZ in left eyes, and diabetes was induced three weeks after injection. A: Each blot is a representative of the results from five rats. B: Occludin levels were semi-quantified by densitometry, and normalized to actin. The rAAV-angiostatin significantly decreased retinal occludin loss as compared to eyes receiving rAAV-lacZ injection at 10 days after induction of diabetes (the asterisk indicates significance using the Wilcoxon signed rank test, n=5, p=0.041).rAAV-angiostatin influence on VEGF expressionOne day after diabetes induction, we observed rAAV-angiostatin-mediated influence of retinal VEGF expression. The retinal VEGF expression in normal control SD rat was 7.56±1.25 pixels. Retinal VEGF was 13.7±3.78 pixels (n=6) in rAAV-lacZ-treated eyes and decreased to 8.69±3.23 pixels (n=6) in rAAV-angiostatin-treated eyes (Figure 6, p=0.043). The retinal VEGF expression in eyes receiving rAAV-lacZ was 11.45±2.73 pixels at 5 days, 9.12±3.12 pixels at 10 days, and 10.41±3.36 pixels at 15 days after induction of diabetes. The retinal VEGF expression in rAAV-angiostatin-treated eyes was 9.52±3.96 pixels at 5 days, 8.31±2.67 pixels at 10 days, and 10.16±3.36 pixels at 15 days after induction of diabetes. There was no statistical difference in retinal VEGF expression in rAAV-lacZ- and rAAV-angiostatin-treated eyes at 5, 10, and 15 days after induction of diabetes.Figure 6The effect of rAAV-angiostatin gene transfer on retinal VEGF expression at 1 day after STZ-induction of diabetes A: The blots show representative of results from six rats. B: VEGF levels were semiquantified by densitometry and normalized by actin levels. rAAV-angiostatin decreased the expression of VEGF as compared to eyes with rAAV-lacZ injection (the asterisk indicates significance using the Wilcoxon signed rank test, n=6, p=0.043).PEDF expressionThere was no significant difference in retinal PEDF expression between eyes exposed to rAAV-angiostatin and rAAV-lacZ at 1, 5, 10, or 15 days after STZ injection (Figure 7A). To further evaluate if rAAV-angiostatin could influence PEDF expression, we infected HUVEC-2C cells with rAAV-angiostatin. No PEDF expression was detected in rAAV-angiostatin-infected HUVEC-2C cells (Figure 7B). Western blot analysis of PEDF plasmid-transfected HUVEC-2C cells was used as a positive control.Figure 7The effect of rAAV-angiostatin gene transfer on retinal PEDF expression A: There was no apparent difference in retinal PEDF expression between eyes exposed to rAAV-angiostatin and rAAV-lacZ. at 1, 5, 10, or 15 days after STZ injection. B: There was no increase in PEDF expression in HUVEC-2C cells receiving rAAVangiostatin compared to control HUVEC-2C cells. Western blotting of the PEDF plasmid-transfected HUVEC-2C cells was used as a positive control.rAAV-angiostatin-mediated downregulation of phospho-p42/p44 MAP kinaseThe retinal phosphor-p42/p44 MAP kinase level of normal control SD rat was 7.03±1.22 pixels. At one day after STZ injection, retinal phosphor-p42/p44 MAP kinase levels were 7.43±0.93 pixels (n=5) in eyes receiving rAAV-lacZ, and was decreased to 4.58±1.36 pixels(n=5) in eyes receiving rAAV-angiostatin (Figure 8, p=0.043). No effect was observed at 5 days after STZ injection (5.64±1.28 pixels for eyes receiving rAAV-lacZ, and 5.44±1.65 pixels for eyes receiving rAAV-angiostatin), 10 days (5.13±0.92 pixels for eyes receiving rAAV-lacZ, and 5.97±1.87 pixels for eyes receiving rAAV-angiostatin) and 15 days (7.37±1.33 pixels for eyes receiving rAAV-lacZ, and 7.25±2.65 pixels for eyes receiving rAAV-angiostatin)Figure 8The effect of rAAV-angiostatin gene transfer on retinal phosphorylation of p42/p44 MAP kinases at 1 day after STZ-based induction of diabetes A: Representative blots show retinal phospho-p42/44 MAP kinase and total p42/44 MAP kinase in normal control eyes and in eyes injected with rAAV-lacZ or rAAV-angiostatin. B: Retinal levels of phosphor-p42/p44 MAP kinase one day after STZ-based induction of diabetes was reduced by rAAV-angiostatin (the asterisk indicates significance using the Wilcoxon signed rank test, n=5, p=0.043).DiscussionRetinal vascular leakage is a major cause of macular edema in diabetic retinopathy and other retinal diseases [1-6]. Traditionally, laser photocoagulation has been used to reduce the vascular leakage induced by diabetes [32,33]. Recently, anti-inflammatory drugs such as triamcinolone [34], or pars-plana vitrectomy with or without internal limiting membrane peeling [35] have been used to attenuate the diabetic macular edema. Due to the duration and severity, an ideal strategy would be to develop an approach involving a single administration of a vector that would result in long-term expression of a suitable therapeutic gene [36].rAAV vectors are highly efficient gene delivery systems which can facilitate long-term transduction [22,23]. We have previously reported in several animal models a gene transfer technique based on the use of rAAV vectors [8,23,25,26,37,38]. This gene transfer technique is particularly attractive for treating ocular disease for reasons of accessibility and long term transduction, and potentially because it would enable clinicians to avoid repeated intravitreal injection [8,37]. We previously reported suppression of laser-induced choroidal neovascularization by an rAAV vector expressing angiostatin [8]. Here, we report that vascular leakage in experimental diabetic rats can be reduced by angiostatin delivery via an rAAV vector. These results suggest that rAAV-angiostatin could be beneficial in the treatment of diabetic macular edema.Angiostatin is a proteolytic fragment of plasminogen and contains kringle domains 1 through 4 [10]. It has been determined that angiostatin is a potent anti-angiogenic factor [11] that can inhibit endothelial cell migration and induce apoptosis in these cells [12]. Recently, intravitreal injection of angiostatin was found to reduce vascular leakage in a rat model of experimental diabetes and in oxygen induced retinopathy [9]. In the same report, the expression of VEGF was found to be downregulated by angiostatin.VEGF is a potent angiogenic factor, expression of which is increased in eyes with diabetic retinopathy [13,14]. VEGF, which is also known as vascular permeability factor (VPF) [39], increases microvascular permeability at very low concentrations [39], and may be important in the pathogenesis of vascular leakage induced by diabetes [40]. Angiostatin-induced reduction of vascular leakage occurs through blockade of VEGF expression [15]. In our study, retinal VEGF expression decreased in eyes receiving rAAV-angiostatin as compared to rAAV-lacZ treated eyes at one and five days after induction of diabetes. Vascular leakage was also decreased in eyes receiving intravitreal injection of rAAV-angiostatin compared to the contralateral eyes receiving rAAV-lacZ injection at 5, 10, and 15 days after induction of diabetes.These results are consistent with previous reports that angiostatin reduces the vascular leakage via blockade of VEGF [13-15,39,40]. How angiostatin reduces vascular leakage through inhibition of VEGF production is still under investigation.It is known that p42/p44 MAP kinase phosphorylation is induced by hypoxia [41]. This phosphorylation is diminished by angiostatin in microvascular endothelial cells [42]. Phosphorylation of p42/p44 MAP kinase promotes VEGF expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex of the VEGF promoter [43]. It is therefore plausible that inhibition of phosphorylation of p42/p44 MAP kinase by angiostatin suppresses VEGF expression under conditions of hypoxia. In our study, retinal phosphorylation of p42/p44 MAP kinase decreased in eyes receiving rAAV-angiostatin at one day after induction of diabetes (Figure 8). Our results in STZ-induced diabetic rats suggest that angiostatin suppresses VEGF expression by inhibition of phosphorylation of the p42/p44 MAP kinase.BRB breakdown is a hallmark of vascular leakage in diabetic retinopathy and other retinal vascular diseases [44,45]. The tight junctions between the retinal vascular endothelial cells constitute an essential structured component of BRB [18]. This barrier limits diffusion of molecules from vessel lumen to the tissue, and thereby maintains the microenvironment of the retina [46]. The barrier protein occludin is decreased in experimental diabetes [21]. VEGF stimulates phosphorylation and redistribution of occludin [19], which is subsequently endocytosed and degraded [47,48]. This process is closely related to the elevated vascular permeability in experimental diabetes [18]. In our study, retinal occludin content in STZ-induced diabetic rats was preserved in eyes receiving rAAV-angiostatin as compared to eyes receiving rAAV-lacZ. The rAAV-angiostatin-induced inhibition of VEGF may therefore suppress vascular leakage by preserving retinal occludin content in STZ-induced diabetic ratsPEDF is a potent angiogenic inhibitor that is counter balanced by the angiogenic effect of VEGF [49-51]. Decreased expression of PEDF in retina is associated with ischemia-induced retinal neovascularization and proliferative diabetic retinopathy [50]. Recently, another proteolytic fragment of plasminogen-kringle 5 (K5) was noted to upregulate PEDF expression in a dose-dependent manner in vascular endothelial cells and in the retina [15]. In our study, there was no statistically significant difference between PEDF expression in eyes receiving rAAV-angiostatin and the contralateral eyes receiving rAAV-lacZ. Our results also revealed that rAAV-angiostatin did not upregulate PEDF expression in HUVEC-2C cells. However, PEDF expression has been shown to be induced at both the mRNA and protein level following injury in the eye [52]. Further research is warranted to explore the effect of rAAV-angiostatin on PEDF expression.Our study showed that transgenic expression of rAAV-angiostatin can reduce retinal vascular leakage in STZ-induced diabetic rats. This effect is associated with downregulation of retinal VEGF and phospho-p42/p44 MAP kinase expression, and a reduction in the retinal occludin loss induced by diabetes. However, the vascular leakage and VEGF expression after induction of diabetes in the SD rat model was demonstrated to be a short-term effect [53]. Gene-based therapies can be as effective and viable as real treatment if long-term expression can be achieved. To demonstrate the long-term effect of rAAV-angiostatin on vascular leakage induced by diabetes, an alternative animal model such as the Brown-Norway rat could be used in a future study.Diabetic macular edema is a major cause of vision loss in diabetic patients [7]. On the basis of these findings, we believe that a similar vector and therapeutic gene could eventually be a useful strategy for long-term preventive or adjunctive therapy for macular edema induced by diabetes. It could serve as the basis for an alternative treatment for patients who are suffering from diabetic macular edema as well as a potentially preventive therapeutic modality for diabetic patients who are at risk for development of macular edema.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2533098",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533098\nAUTHORS: D William Provance, Erin J Addison, Patrick R Wood, David Z Chen, Colleen M Silan, John A Mercer\n\nABSTRACT:\nBackgroundMyosin-Vb has been shown to be involved in the recycling of diverse proteins in multiple cell types. Studies on transferrin trafficking in HeLa cells using a dominant-negative myosin-Vb tail fragment suggested that myosin-Vb was required for recycling from perinuclear compartments to the plasma membrane. However, chemical-genetic, dominant-negative experiments, in which myosin-Vb was specifically induced to bind to actin, suggested that the initial hypothesis was incorrect both in its site and mode of myosin-Vb action. Instead, the chemical-genetic data suggested that myosin-Vb functions in the actin-rich periphery as a dynamic tether on peripheral endosomes, retarding transferrin transport to perinuclear compartments.ResultsIn this study, we employed both approaches, with the addition of overexpression of full-length wild-type myosin-Vb and switching the order of myosin-Vb inhibition and transferrin loading, to distinguish between these hypotheses. Overexpression of full-length myosin-Vb produced large peripheral endosomes. Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition. Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1).ConclusionAll results favored the peripheral dynamic tethering hypothesis.\n\nBODY:\nBackgroundMolecular motors generally are thought to be recruited to vesicles or organelles to provide directional movement; however, this perspective is complicated by evidence showing that multiple motors, using multiple cytoskeletal substrates, are found on individual vesicles and organelles [1-7]. While kinesins and dyneins clearly transport cargo for long distances in vivo, there is surprisingly little evidence for such a role for unconventional myosins in higher eukaryotes [8]. Biophysical studies have shown that many myosin head domains bind more tightly to actin in response to loading [9-12], but these adaptations usually are interpreted as promoting processive transport of cargo over long distances. However, the biophysical data also are consistent with adaptation to function as dynamic tethers or tensioners between actin filaments and other cytoplasmic structures. In this context, tethering is distinct from docking, in that it may simply represent a net balance of forces, movements, and/or positions. Accordingly, retention (and active transport) within cortical actin might prevent endosomes from encountering microtubules, so tethering may represent an effect rather than a distinct molecular mechanism. Actual point-to-point transport of cargo by unconventional myosins in a cellular context might be relatively rare; for example, to reposition the myosin in the absence or reduction of load, halting as a new load is sensed.Myosin-Vb, originally named myr 6 [13], is a member of one of the most ancient divisions of the myosin superfamily [14], with diverse cellular functions. It interacts with the brain-expressed RING finger protein BERP, Rab11a, Rab11a-FIP2, Rab11b, Rab25, and Rab8a [15-19]. It has been implicated in recycling of transferrin and its receptor [16,18,20], the chemokine receptor CXC2 [21], HIV Vpu [22], acetylcholine receptors [23], the polymeric IgA receptor [16,24], and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor subunit GluR1 [25]. It also has been implicated in formation of bile canaliculi [26].Overexpression of tail fragments of unconventional myosins has been the standard technique for their inhibition, and data from these experiments are usually interpreted in the context of point-to-point transport. For myosin-Vb in transferrin trafficking, overexpression of a tail fragment in HeLa cells caused accumulation of transferrin in perinuclear compartments, suggesting that myosin-Vb functions in the transport of vesicles between perinuclear recycling endosomes and the plasma membrane [16]. By contrast, we adapted a chemical-genetic method pioneered by Shokat and colleagues for kinases [27,28] to unconventional myosins, allowing us to acutely and specifically induce tight binding of a sensitized mutant myosin to actin by microinjection or dialysis of an ADP analog [29-32]. When we inhibited the sensitized mutant myosin-Vb (also dominant-negative inhibition), it prevented accumulation of transferrin-positive vesicles and organelles in the perinuclear region [20]. This result was inconsistent with the transport hypothesis, because if myosin-Vb is required for transport between perinuclear compartments and the plasma membrane, induction of tight binding to actin should have caused transferrin to accumulate in perinuclear compartments.These apparently contradictory results could be reconciled if myosin-Vb acts peripherally as a dynamic tether that antagonizes the retrograde transport of transferrin to perinuclear compartments, possibly by holding the parental organelle in the periphery during fission. We also observed an increase in plasma-membrane transferrin receptor upon myosin-Vb inhibition [20], suggesting that chemical-genetic inhibition had shunted trafficking to the rapid peripheral pathway [33]. The tail-fragment overexpression data can be explained as release of the peripheral endocytic compartments from actin, allowing entire peripheral endosomes to be transported to the perinuclear region.Our hypothesis is illustrated in Fig. 1. Peripheral endosomes are retained in the periphery by multiple myosin-Vb motors whose heads periodically detach from actin (green) as they go through the ATPase cycle, but usually rapidly reattach, as suggested by biophysical data (Fig. 1A). In Fig. 1B, dynein (or a minus-end-directed kinesin; its head domain is shown as the letter \"D\") attaches to a microtubule and exerts retrograde force. Occasionally, adjacent myosin-Vb detaches from actin (dotted circle) and cannot reattach because dynein has pulled it away from the actin filament. The remaining myosins hold the bulk of the endosome in place. In Fig. 1C, fission has occurred, and the daughter vesicle moves retrogradely; after the switch to microtubules, myosin-Vb is carried along as a passenger. Although we hypothesize that myosin-Vb primarily functions as a dynamic tether, our model does not preclude myosin-Vb-dependent meandering within the peripheral actin network. We have diagrammed two different means of binding myosin-Vb to the endosome as cyan and purple circles, since two different means have been demonstrated experimentally: Rab11a [16] and the CART complex [18].Figure 1Model and normal distributions of myosin-Vb and transferrin. (A) Peripheral endosomes are retained in the periphery by multiple myosin-Vb motors whose heads periodically detach from actin as they go through the ATPase cycle, but usually reattaching. (B) Dynein (or a minus-end-directed kinesin) attaches to a microtubule and exerts retrograde force. Occasionally, adjacent myosin-Vb detaches from actin (dotted circle), allowing dynein to pull it away from the actin filament. (C) Following fission, the daughter vesicle moves retrogradely, carrying myosin-Vb as a passenger. (D, E, F) In HeLa cells expressing low levels of eGFP-myosin-Vb, colocalization between myosin-Vb (green) and transferrin (red) is rare (arrow) and transient (also see Additional file 1).In this study, we have employed three different perturbations of myosin-Vb function to further test the dynamic tethering hypothesis, which makes clear predictions: first, overexpression of full-length, functional myosin-Vb will prevent transferrin from reaching perinuclear compartments; second, chemical-genetic inhibition of sensitized mutant myosin-Vb in cells after transferrin loading (in our previous study, it was done before transferrin loading) will neither cause accumulation of transferrin in perinuclear compartments nor prevent transferrin from moving from perinuclear compartments to the plasma membrane; and third, overexpression of the myosin-Vb tail fragment will cause at least some peripheral endocytic markers to assume more perinuclear distributions. The new data generally contradict the transport hypothesis. In addition, our data suggest that members of the myosin-V family may play a ubiquitous function in modulating vesicle transport along microtubules, as they are available to interact with passing actin filaments as passengers. Applied more broadly, our data suggest that identifying endocytic compartments by their positions within the cytoplasm may be unreliable in the context of significant experimental disruptions.Results and DiscussionWe have shown that expression of low levels of exogenous myosin-Vb (25–40% of endogenous levels) does not alter the trafficking of transferrin [20]. However, the dynamic tethering hypothesis predicts that exceeding endogenous levels with wild-type exogenous myosin-Vb will alter the balance of forces, reducing the extent and/or rate of retrograde movement from peripheral to perinuclear compartments. To test this prediction, we increased the amount of myosin-Vb associated with those compartments by transiently transfecting HeLa cells with a full-length, wild-type myosin-Vb construct. To allow imaging of live cells, we used a construct with an N-terminal eGFP tag [16]. We compared the distribution of eGFP-tagged myosin-Vb with that of our C-terminal -tagged (V5 and 6x-His) version [20], and observed no significant differences (data not shown). At low levels of eGFP-myosin-Vb expression, we observed only occasional, highly dynamic, colocalization of myosin-Vb and transferrin (arrows, Fig. 1D,E,F; Additional file 1).Figure 2 and Additional files 2, 3, 4, 5, 6, 7, 8 show transferrin accumulation in peripheral compartments as a function of the overexpression level of eGFP-myosin-Vb, which the dynamic tethering hypothesis predicts will cause the coalescence and caging of peripheral endosomes by actin (Fig. 2A). A coalescence of actin around the enlarged peripheral endosomes is shown by the colocalization of myosin-Vb and actin (Fig. 2B,C).Figure 2Overexpression of full-length, wild-type eGFP-myosin-Vb causes coalescence of peripheral endocytic compartments and inhibits perinuclear accumulation of transferrin. HeLa cells were transiently transfected with full-length, wild-type myosin-Vb tagged with eGFP and imaged 24 h after transfection. (A) Diagram depicting predicted results. (B, C) Colocalization of actin on enlarged compartments (arrows) with eGFP-myosin-Vb. (D, E, F) In cells expressing high levels of eGFP-myosin-Vb coincident with exposure to transferrin (arrows), large, peripheral organelles decorated with myosin-Vb also contain transferrin. (G, H, I, J, K, L) In cells exposed to a 1-min pulse of transferrin 24 h after transfection and 10 min before imaging, transfected cells (arrows) contain large, peripheral organelles decorated with myosin-Vb that lack transferrin. Cells expressing lower levels of myosin-Vb (arrowheads, panel K; too low to be seen in panel J) accumulate less transferrin than the surrounding untransfected cells (arrowheads). Bar, 15 μm.HeLa cells that endocytosed fluorescent transferrin before and during overexpression of eGFP-myosin-Vb sequestered transferrin in large peripheral compartments decorated with myosin-Vb (Fig. 2D,E,F; Additional file 2), suggesting that fission of the compartments in which myosin-Vb and transferrin normally transiently colocalize (arrow, Fig. 1D,E,F; Additional file 1) was inhibited. By contrast, when transferrin was introduced after overexpression of myosin-Vb, transferrin was not colocalized with myosin-Vb in the enlarged peripheral compartments (arrows, Fig. 2F–K; Additional files 3, 4, 5). In addition, transferrin failed to accumulate in perinuclear compartments. As a negative control, we expressed a truncated myosin-Vb consisting of the head domain and first IQ domain, which had no effect on transferrin localization (data not shown). These data suggest that that overexpression of myosin-Vb prevents transferrin from both entering into and exiting from a normally dynamic, short-lived endocytic compartment.In isolation, the static images shown in Figure 2 can be fit to the anterograde transport model if overexpression caused rapid transport of transferrin from perinuclear compartments while delaying its passage through cortical actin. However, Additional files 3, 4, 5 show that transferrin is not reaching perinuclear compartments.As these data suggest that fission of vesicles from peripheral endocytic compartments and/or their transport to perinuclear compartments had been prevented by increased tethering to cortical actin, we examined the distribution of the endocytic markers Rab11a, Rab4, and Rab5. Cotransfections with eGFP-myosin-Vb and the recycling endosome marker mRFP-Rab11a showed virtually complete colocalization at high levels of myosin-Vb expression (Additional file 6). By contrast, little colocalization was observed in cells cotransfected with eGFP-myosin-Vb and the early endosome markers mRFP-Rab4 (Additional file 7) and mRFP-Rab5 (Additional file 8), suggesting that trafficking through early endosomes was not prevented. The videos also show that the enlarged endosomes are relatively static, consistent with increased tethering forces and caging by actin.In a previous study, we used a chemical-genetic approach to show that induction of tight binding of sensitized myosin-Vb to actin, before addition of transferrin, prevented transferrin from accumulating in perinuclear compartments [20]. Our hypothesis is diagrammed in Fig. 3A, and the effect of inhibition before transferrin uptake, demonstrated previously, is shown in Fig. 3B. If myosin-Vb is required for transport from perinuclear compartments to the plasma membrane, then inducing tight binding of myosin-Vb to actin after transferrin loading should increase transferrin accumulation in perinuclear compartments, just as myosin-Vb tail overexpression does. We therefore transfected HeLa cells with Y119G sensitized mutant (Fig. 3) and wild-type control (not shown) myosin-Vb, loaded them with fluorescent transferrin, and microinjected the specific inhibitor of Y119G myosin-Vb, N6-(2-phenylethyl)-ADP (PE-ADP) [20]. Only cells with a punctate eGFP localization, representing lower expression levels, were chosen for microinjection. When PE-ADP was injected 10 min (data not shown) and 30 min (Fig. 3D,E,F) following the addition of transferrin, we still observed a decrease in fluorescence intensity in the perinuclear region of the transfected and injected cells (Fig. 3D,E,F) as well as rapid movement of transferrin when it did not colocalize with myosin-Vb (Additional file 9). These data, as well as the limited colocalization between transferrin and myosin-Vb, indicate that myosin-Vb activity is not required to transport transferrin from perinuclear compartments to the plasma membrane. These data are much more consistent with the peripheral tethering hypothesis, because the peripheral site of myosin-Vb function has been bypassed by loading with transferrin before induction of tight binding of myosin-Vb to actin.Figure 3Inhibition of myosin-Vb after loading with transferrin does not prevent transit from perinuclear recycling endosomes. HeLa cells transiently expressing sensitized myosin-Vb were loaded with Alexa 546-transferrin, washed, incubated in growth medium for 30 min, and imaged for myosin-Vb and transferrin. (A) Diagram depicting predicted results; the sensitized mutant myosin-Vb is shown in red and PE-ADP is shown as a green circle. (B) Inhibition of accumulation of transferrin (red) added after myosin-Vb inhibition by microinjection of PE-ADP2. Injected cells have blue nuclei. (C, D, E, F, G, H) The cell expressing sensitized myosin-Vb (center, panel C) was immediately injected with PE-ADP and the same field was imaged 30 min later (F, G, H). Panels C and D are overlaid in panel E, and panels F and G are overlaid in panel H. Bar, 15 μm.While the inhibition of the Y119G sensitized mutant myosin-Vb in preloaded cells did not cause transferrin accumulation in perinuclear compartments, the data were not as simple as they were predicted to be by the dynamic tethering hypothesis, as myosin-Vb inhibition retarded the depletion of transferrin from perinuclear compartments relative to control cells (Fig. 3G,H). Upon closer examination, our induction of binding of myosin-Vb to actin had the general effect of halting nearly all motion of myosin-Vb-decorated structures within the cell (Fig. 4A). The motility of eGFP-myosin-Vb before and after microinjection was analyzed using kymographs (Fig. 4B,C for the cells shown in Fig. 4A, Fig. 4D,E for additional negative control cells; also see Additional files 10, 11, 12). Binned measurements of instantaneous particle speeds in the presence and absence of PE-ADP (Fig. 4F, Additional files 10 and 11) show that not only was slower actin-based motility (0.15 – 0.3 μm/s) inhibited, but higher-speed movements of myosin-Vb-decorated particles caused by microtubule-based motors (> 0.7 μm/s) were halted as well. No such inhibition was observed under control conditions, which included cells expressing Y119G myosin-Vb after injection of vehicle plus fluorescent Dextran without PE-ADP (data not shown), as well as cells expressing wild-type myosin-Vb after PE-ADP injection (Fig. 4G, Additional file 12).Figure 4Chemical-genetic inhibition of myosin-Vb halts myosin-Vb-decorated particles, including those being transported via microtubules. (A) Representative image of two HeLa cells expressing sensitized Y119G mutant eGFP-myosin-Vb before injection of the upper cell with PE-ADP. Bar, 15 μm. (B and C) Kymographs from the cells shown in panel A (y axes represent lines wx and yz from panel A). (D and E) Kymographs from additional negative control cells expressing wild-type myosin-Vb injected with PE-ADP and wild-type myosin-Vb injected with dextran respectively. (F and G) Histograms of instantaneous speeds of myosin-Vb-labeled vesicles before (black bars) and after (white bars) PE-ADP injection in cells expressing wild-type and Y119G mutant eGFP-myosin-Vb respectively; Speeds were measured for 1178 (before injection) and 621 particles (after) for panel F, and 717 and 551 respectively for panel G. (H) Instantaneous speeds of wild-type eGFP-myosin-Vb-labeled vesicles before (black bars) and after (white bars) depolymerization of actin by latrunculin A; the Y119G mutant gave indistinguishable results (data not shown). Speeds were measured for 707 (before) and 206 (after) particles. (I and J) Diagrams depicting additions to the dynamic tethering hypothesis to accommodate these data. Kinesin is represented with the letter \"k\" for the head domain.The arrest of microtubule-based motility of myosin-Vb-decorated particles was unexpected, and we initially suspected that it might have been an artifact of high effective ADP concentration in the form of the microinjected PE-ADP analog. To test the hypothesis that myosin-Vb interacts transiently with actin filaments during microtubule-based transport under normal conditions, we measured the speeds of particles decorated with wild-type eGFP-tagged myosin-Vb before and after the addition of latrunculin A. If myosin-Vb (or other myosins) normally interacts with actin filaments, latrunculin A treatment should increase both mean speed and the proportion of vesicles moving at 0.7–1.0 μm/sec. This prediction was confirmed, as latrunculin treatment nearly doubled the proportion of particles exhibiting rapid movement (Figure 4H), in contrast with results from melanosome transport in fish melanophores [34]. The modification of the dynamic tethering hypothesis to account for these data is diagrammed in Fig. 4I and 4J.The dynamic tethering hypothesis further predicts that some markers found in peripheral endocytic compartments are likely to be shifted to a more perinuclear distribution by myosin-Vb tail overexpression (Fig. 5A). We tested this prediction for early endosomal antigen-1 (EEA1), which had a dispersed pattern in control cells (Fig. 5B,C,D, arrowhead), while in cells expressing the eGFP/myosin-Vb tail chimera [16], EEA1 was much more concentrated, in an asymmetric pattern primarily on one side of the nucleus (Fig. 5B,C,D, arrows).Figure 5eGFP-myosin-Vb tail overexpression displaces EEA1 and Rab11a to more perinuclear positions. HeLa cells were transfected with the eGFP-myosin-Vb tail construct and allowed to express overnight. (A) Diagram depicting displacement of peripheral endosomes. (B) Immunofluorescent detection of EEA1. (E, F, G) Cotransfection with the mRFP-Rab11a construct. (H, I, J) Cells cotransfected with the mRFP-Rab8a construct. (C, F, I) eGFP-myosin-Vb tail. (D, G, J) overlays of B+C, E+F, and H+I respectively; arrows, cells expressing the myosin-Vb tail fragment; arrowheads, control cells not expressing the tail. Bar, 15 μm.Based on the change in distribution of EEA1 coupled with its failure to colocalize with the myosin-Vb tail, we hypothesize that in the presence of the tail, endosomes still are transported to more perinuclear regions of the cytoplasm, but the fission between their domains that normally occurs in peripheral regions occurs in a more perinuclear location. We then confirmed the effect of the myosin-Vb tail on Rab11a redistribution. As observed by Lapierre et al., the dispersed pattern observed in untransfected control cells (Fig. 5E,F,G, arrowheads) was changed to a more perinuclear pattern by overexpression of the eGFP/myosin-Vb tail (arrows).We next examined Rab8a, which has been shown to interact in vitro with myosin-Vb [19]. We observed a nearly normal distribution of Rab8a despite the overexpression of the myosin-Vb tail (Fig. 5H,I,J). These results are consistent with the differences between Rab11a and Rab8a compartments and pathways observed by Roland et al., and indicate that the affinity of the myosin-Vb tail domain for Rab8a is much lower than its affinity for Rab11a. This result also is consistent with their inability to observe interaction between myosin-Vb and Rab8a in a cellular context.Since mosaic endosomes have been observed with every possible combination of Rab4, Rab5, and Rab11a [35], we examined Rab4 and Rab5 distribution. Overexpression of the myosin-Vb tail produced a slight alteration in the distribution of Rab4 (Fig. 6A,B,C,D), but no significant effect on Rab5 distribution (Fig. 6F,G,H), which is puzzling given the association between EEA1 and Rab5 [36].Figure 6(A-H) Overexpression of eGFP-myosin-Vb tail causes a slight shift in Rab4 distribution, but has little effect on Rab5 distribution. HeLa cells were transiently transfected with eGFP-myosin-Vb tail fragment (B,C,D,F,G,H) and mRFP-Rab4 (A,B,C,D) or mRFP-Rab5 (E,F,G,H) and imaged after overnight incubation. Bar, 15 μm.To summarize our model, myosin-Vb is associated with multiple compartments, of which only some are involved in transferrin trafficking. Myosin-Vb primarily tethers a subset of peripheral, Rab11a-positive endocytic compartments to cortical actin, opposing forces from dynein or minus-end-directed kinesins and retaining the compartment in the actin-rich periphery. This is analogous to the mechanism of Velcro™, except that instead of hooks bending, the myosin-Vb heads are going through the ATPase cycle and periodically releasing from actin. In this analogy, overexpression of full-length, wild-type myosin-Vb (Fig. 2) causes greater retention of normally endocytic compartments in the periphery, leading to their coalescence, because the increase in number of myosins outweighs their individual cycling off and back onto actin. These data strongly suggest that while this compartment is normally rare and transient (Fig. 1), all transferrin still must pass through it to reach perinuclear compartments. Both our overexpression of myosin-Vb and overexpression of the myosin-Vb tail create artifacts. In the former case (Fig. 2), caging by actin causes coalescence and blockage of both entry and exit; while in the latter case [16], release from actin causes what we believe to be virtually the same compartment to collapse to a perinuclear location. Chemical-genetic inhibition is analogous to preventing individual Velcro™ hooks from bending, but does not prevent entry and exit into this compartment via the peripheral pathway [20].Myosin-Va, the founding member of this myosin family, appears to have a similar function, albeit involving different compartments. In melanocytes, we first suggested a peripheral tethering function for myosin-Va based on the mutant phenotype, a perinuclear accumulation of melanosomes [37]. The best-characterized system for melanosome transport has been Xenopus melanophores [4,5,38,39], with similar, but less dynamic, results from murine melanocytes [40]. In both cases, myosin-Va function was hypothesized to provide not only peripheral capture, but transport within the periphery as well. While pauses in microtubule-based movement attributed to myosin-Va have been observed [41,42], these studies represent the first such observation for myosin-Vb. In general, myosin-Vb appears to perform the same function in the endocytic pathway as myosin-Va performs in exocytic pathways, and our future experiments will test the validity of our generalization. In a technical context, our results suggest that membrane compartments cannot necessarily be reliably identified by their locations within the cytoplasm in cells in which trafficking has been grossly perturbed by manipulation, particularly overexpression, of any relevant component.MethodsExpression ConstructsThe full-length eGFP-wild-type myosin-Vb, eGFP-myosin-Vb tail, mRFP-Rab4a, mRFP-Rab5a, mRFP-Rab8a and mRFP-Rab11a expression constructs were gifts from Jim Goldenring and are based on the peGFP-C2 expression vector (Clontech). The sensitized Y119G mutant eGFP myosin-Vb was created by shuttling a 989-bp ClaI/BstEII fragment from pEcho/pcDNA3.1 Y119G myosin-Vb [20]. The control eGFP-tagged myosin-Vb 1IQ expression vector was created by amplifying the eGFP-tagged, wild-type myosin-Vb sequence for the head domain through the first IQ domain and cloning into pYY8.Cells and transfectionHeLa cells were cultured as described [20]. For transfections, 1 μg of Lipofectamine 2000 (Invitrogen) was added to 40 μl of OptiMEM (Invitrogen), then mixed with 0.5 μg of DNA diluted into 40 μl of OptiMEM according to manufacturer's instructions. HeLa cells were trypsinized, collected and resuspended in complete medium at a concentration of 1 × 106 cells/ml. The cell suspension (80 μl) was added to the DNA/liposome mixture and plated as 40-μl dots in live-cell chambers (Bioptechs, Butler, PA) or glass coverslips, incubated for 1–2 h and then flooded with complete medium. Cells were used in experiments 24–48 h after transfection.ImmunofluorescenceEEA1 was detected using a monoclonal antibody (BD Transduction Laboratories 610456). The primary antibody was detected with Alexa-546- or Alexa-647-labeled goat anti-mouse secondary (Invitrogen). For cell outlines, actin was stained with Alexa-647-labeled phallacidin (Invitrogen).Transferrin trafficking, microinjection, and latrunculin treatmentTransfected cells were incubated in serum-free medium for 60 min, then exposed to 10 μg/ml Alexa 546-labeled transferrin (Invitrogen) for 1 min, washed 3 times with PBS and incubated in pre-equilibrated complete medium for the duration of live-cell experiments. For concomitant labeling with transferrin before expression of exogenous eGFP-myosin-Vb (Fig. 2D,E,F), fluorescent transferrin (10 μg/ml) was added to the complete medium used to flood the coverslips during transfection as described above. During live-cell experiments, stage positions of individual transfected cells were stored using MetaMorph (Molecular Devices) and a motorized stage (Prior). To inhibit Y119G myosin-Vb, HeLa cells were injected in the nucleus with 10 mM PE-ADP, 100 mM KCl, 8 mM K-phosphate (pH 7.0), 0.05 mg/ml fixable Alexa 647-labeled dextran (Invitrogen), and 10 mM Mg-ATP using a Harvard Apparatus PLI-100 at 8–15 kilopascals; negative control injections contained the same solution lacking PE-ADP. Assuming an injection volume of ~3% cell volume, the final concentration of PE-ADP was ~300 μM. To inhibit actin polymerization, latrunculin A (Molecular Probes) was added to medium at 2.5 μg/ml.Imaging and QuantitationAll images were obtained using a Nikon TE2000E equipped with a Q57 12-bit CCD camera (Roper Scientific) controlled by MetaMorph software. Images were obtained through a 60× (1.2 NA) water-immersion lens that was maintained at 37°C using an objective heater (Bioptechs). For time-lapse movies, images were obtained at a rate of 1 frame/sec over a 1-min time course. Fluorescence imaging of the dextran to identify injected cells was performed following the time-lapse imaging. Instantaneous speeds of individual particles were measured by observers (blindly with respect to experimental conditions) using the Track Points package of MetaMorph and the data were exported to Microsoft Excel. Measurements were obtained from 5–10 cells per condition, and numbers of particles are provided in the Figure 3 legend. Since pixel size produced submicron/sec speed measurement errors, speeds were separated into 3 bins: 0–0.15 μm/sec (stationary and actin-based), 0.16–0.70 μm/sec (both actin- and microtubule-based), and 0.71–1.0 μm/sec (microtubule-based).Additional filesAll videos are of HeLa cells.Abbreviations ListEEA1: early endosomal antigen-1; and PE-ADP: N6-(2-phenylethyl)-ADP.Authors' contributionsDWP performed most of the experiments, performed most of the data analysis, and designed the project. EJA, PRW, and DZC analyzed particle speeds and assisted in experiments as summer research interns. CMS assisted in performing experiments. JAM designed the project with DWP, performed data analysis, and wrote the manuscript.Supplementary MaterialAdditional file 1Low-level expression of full-length eGFP-myosin-Vb (green) shows rare, dynamic colocalization (circles) of myosin-Vb and transferrin (red); same cell as shown in Figure 1D–F. Frame acquisition rate, 0.5/sec; elapsed seconds are displayed at lower right.Click here for fileAdditional file 2Overexpression of full-length eGFP-myosin-Vb (green) in the presence of transferrin (red; added at the time of transfection) produces enlarged, less-motile peripheral endosomes decorated with myosin-Vb and containing transferrin at 24 h post transfection; same cells as shown in Figure 2D–F. Frame acquisition rate, 0.5/sec; elapsed seconds are displayed at lower right.Click here for fileAdditional file 3Overexpression of full-length eGFP-myosin-Vb produces enlarged, less-motile peripheral endosomes decorated with myosin-Vb; same field as shown in Figure 2J–L. Frame acquisition rate, 0.5/sec; frame display rate, 3/sec.Click here for fileAdditional file 4Overexpression of full-length eGFP-myosin-Vb (not shown) prevents entry of Alexa 546-labeled transferrin (shown) into perinuclear compartments; same field as Additional file 3. Frame acquisition rate, 0.5/sec; frame display rate, 3/sec.Click here for fileAdditional file 5Overlay of Additional file 3 (myosin-Vb, green) and Additional file 4 (transferrin, red).Click here for fileAdditional file 6Rab11a (red) colocalizes with eGFP-myosin-Vb (green) at high myosin-Vb expression levels. Frame acquisition rate, 0.5/sec; frame display rate, 6/sec.Click here for fileAdditional file 7Rab4 (red) does not colocalize with eGFP-myosin-Vb (green) at high myosin-Vb expression levels. Frame acquisition rate, 0.5/sec; frame display rate, 6/sec.Click here for fileAdditional file 8Rab5 (red) does not colocalize with eGFP-myosin-Vb (green) at high myosin-Vb expression levels. Frame acquisition rate, 0.5/sec; frame display rate, 6/sec.Click here for fileAdditional file 9Chemical-genetic inhibition of sensitized mutant (Y119G) eGFP-myosin-Vb by PE-ADP microinjection does not prevent movement of transferrin-positive particles. Cells were loaded with fluorescent transferrin (red) 30 min before myosin-Vb was inhibited in the center cell by PE-ADP. Frame acquisition rate, 1/sec; frame display rate, 3/sec.Click here for fileAdditional file 10Chemical-genetic inhibition of sensitized mutant (Y119G) eGFP-myosin-Vb by PE-ADP microinjection (cell on left) halts movement of all myosin-Vb-decorated particles, including those being transported via microtubules; same field as Figure 3A. Uninjected control cell is on the right. Frame acquisition rate, 1/sec; frame display rate, 10/sec.Click here for fileAdditional file 11Same conditions as Additional file 10 without a control uninjected cell. Frame acquisition rate, 1/sec; frame display rate, 10/sec.Click here for fileAdditional file 12Negative control cell expressing wild-type eGFP-myosin-Vb; PE-ADP injection (immediately before imaging) does not halt movement of myosin-Vb-decorated particles. Frame acquisition rate, 1/sec; frame display rate, 10/sec.Click here for file\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2533307.json

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+{
+    "id": "PMC2533307",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533307\nAUTHORS: Frank Dressler, Cornelia Wermes, Eckart Schirg, Angelika Thon\n\nABSTRACT:\nThree pediatric patients with different illnesses leading to knee arthritis and large Baker cysts and additional calf swelling are reported. Calf swelling was due to true popliteal venous thrombosis and not to the much more common cause of pseudothrombophlebitis. Careful ultrasound examination can differentiate these two causes of calf swelling. Even though all our patients had risk factors for thrombophilia, we do not recommend routine thrombophilia work-up for all arthritis patients in the absence of thrombosis.\n\nBODY:\nLarge Baker cysts-particularly following rupture-may lead to calf swelling and pseudothrombophlebitis that has been described in many adult and juvenile patients [1,2]. Pseudothrombophlebitis implies that symptoms of a venous thrombosis such as calf swelling are present, but not caused by thrombophlebitis. True popliteal venous thrombosis has only rarely been reported [3]. We recently saw 3 patients with different forms of juvenile arthritis and Baker cysts without a prior personal or family history of thrombotic events who developed popliteal venous thrombosis and upon work-up following the thrombosis had risk factors for thrombophilia.Patient 1 was a 13-year old boy with a 9-year history of systemic juvenile idiopathic arthritis who developed left knee arthritis after a period of 18 months of disease remission off any medication. Several days later he also complained of painful calf swelling. On ultrasound examination a Baker cyst (52 × 28 × 24 mm) and 2 smaller cysts were found as well as incomplete popliteal venous thrombosis. During treatment with enoxaparin the thrombus dissolved within 2 weeks, and enoxaparin was stopped after 5 months. The knee arthritis improved with naproxen and methotrexate and after intraarticular injection of triamcinolone hexacetonide. At last follow-up 18 months later, the patient felt entirely well without effusion but a very small cyst remained.Patient 2 was a 9-year-old girl with juvenile idiopathic oligoarthritis and 5 months of right knee arthritis treated with naproxen who newly complained of painful calf swelling. Sonography showed a 74 × 34 × 13 mm large Baker cyst as well as an incomplete thrombosis of the popliteal vein. With enoxaparin normal venous flow was reestablished within 3 months and enoxaparin was then stopped. The arthritis improved rapidly following intraarticular injection of triamcinolone hexacetonide and continued naproxen. Nine months later the girl had no complaints, and sonography found a smaller Baker cyst and a very small effusion.Patient 3 was a 14-year old boy initially seen at another hospital with right knee arthritis. He was HLA-B27-positive and had highly positive IgG antibody titers against Borrelia burgdorferi. During 2 weeks of intravenous ceftriaxone his arthritis disappeared, but a few days after the end of antibiotic therapy he developed contralateral knee arthritis and calf swelling. Sonography found a large Baker cyst (86 × 12 mm) and complete popliteal venous thrombosis. He was treated with continuous intravenous heparin and switched to phenprocoumone within a few days, and his thrombosis disappeared within 6 weeks. He also received naproxen. When we first saw the patient 6 weeks after onset of the thrombosis, his arthritis had markedly improved, and the cyst could no longer be seen on ultrasound. Phenprocoumone and naproxen were both stopped 3 months after the onset of thrombosis, and another 13 months later the patient was asymptomatic and without evidence of joint effusion, cyst or thrombosis.A broad investigation for risk factors for thrombophilia was performed in all 3 patients and risk factors found in our patients are shown in Table 1. Each patient had several risk factors. In particular, patient 2 had 2 known risk factors for venous thrombosis with heterozygous mutations in the prothrombin and the factor V genes. Other risk factors such as the PAI-1 (plasminogen activator inhibitor) and fibrinogen polymorphisms have been shown to increase the risks of stroke or myocardial infarction, but may have contributed to the thrombotic events in our patients. More common risk factors such as factor V Leiden, deficiencies in proteins C or S or antiphospholipid-antibodies were not present in our patients. The thrombophilia risk factors in our patients have not led us to restrict their physical activitites.Table 1Thrombophilia risk factors present (+) or absent (-) in our patients. Only those risk factors present in at least one of our patients are shown.Patient 1Patient 2Patient 3Prothrombin G20210A mutation-+-Factor V A4070G mutation*-+-Lipoprotein (a) increase++-Methylenetetrahydrofolatereductase (MTHFR) mutation**+++PAI-1 4G/4G mutation+-+β fibrinogen G455A mutation+++Lupus antibody2+--Factor VIII increase3-++All mutations were heterozygous except for the homozygous MTHFR mutation A1298C in patient 1.*This is not the factor V Leiden mutation.**MTHFR mutations were homozygous A1298C in patient 1, heterozygous A1298C in patient 2 and C677T in patient 3.PAI = Plasminogen Activator Inhibitor.2Lupus antibody was an IgM against beta-2-glycoprotein.3Factor VIII increases were around 200% of the expected and present at the time of thrombosis, but may also be explained as acute phase reaction.In conclusion, large Baker cysts can be mechanical factors contributing to popliteal vein thrombosis, at least in patients with risk factors for thrombophilia. Patients with large Baker cysts and calf swelling should receive careful ultrasound evaluation for the possibility of true thrombosis that could have been mistaken as pseudothrombophlebitis. Since thrombosis remains rare in patients with Baker cysts, we do not recommend routine thrombophilia work-up in the absence of thrombosis.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsFD and AT were the clinicians in charge of pediatric rheumatology patient care, CW performed the thrombophilia work-ups, ES did the ultrasound studies. All authors contributed to writing the manuscript with FD as the principal author.ConsentWritten informed consent was obtained from the patient or where applicable the patient's parents, for publication of this Case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2533336.json

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+{
+    "id": "PMC2533336",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533336\nAUTHORS: Daibin Zhong, Yaw Afrane, Andrew Githeko, Liwang Cui, David M Menge, Guiyun Yan\n\nABSTRACT:\nBackgroundSince the late 1980s a series of malaria epidemics has occurred in western Kenya highlands. Among the possible factors that may contribute to the highland malaria epidemics, parasite resistance to antimalarials has not been well investigated.MethodsUsing parasites from highland and lowland areas of western Kenya, we examined key mutations associated with Plasmodium falciparum resistance to sulfadoxine – pyrimethamine and chloroquine, including dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps), chloroquine resistance transporter gene (pfcrt), and multi-drug resistance gene 1 (pfmdr1).ResultsWe found that >70% of samples harbored 76T pfcrt mutations and over 80% of samples harbored quintuple mutations (51I/59R/108N pfdhfr and 437G/540E pfdhps) in both highland and lowland samples. Further, we did not detect significant difference in the frequencies of these mutations between symptomatic and asymptomatic malaria volunteers, and between highland and lowland samples.ConclusionThese findings suggest that drug resistance of malaria parasites in the highlands could be contributed by the mutations and their high frequencies as found in the lowland. The results are discussed in terms of the role of drug resistance as a driving force for malaria outbreaks in the highlands.\n\nBODY:\nBackgroundMalaria is a major public health problem in sub-Saharan Africa and Plasmodium falciparum infection is a leading cause of morbidity and mortality inflicting a huge economic burden in countries where the disease is endemic [1]. It is estimated that death toll of malaria exceeds one million people each year, and the victims are primarily children under the age of five [2]. Until the early 1980s, the African highlands (generally referred to areas of >1,500 m above sea level) were either free of malaria or had very low incidences of the disease; however, since the late 1980s a series of malaria epidemics has occurred [3-9]. Among the many factors that may contribute to the highland malaria epidemics, resistance of the parasites to multiple antimalarials has not been extensively investigated. Resistance to antimalarial drugs is one of the major obstacles for effective malaria control. The first case of chloroquine (CQ) resistance in Kenya was reported in 1977 [10]. In 1993, resistance levels had reached 70% [11]. In 1998, the Ministry of Health of Kenya changed the first line of treatment from chloroquine to sulfadoxine – pyrimethamine (SP; Fansidar®) [12]. In 2004, the Ministry of Health of Kenya officially changed the first-line drug to artemether/lumefantrin (Coartem™) [13].Drug resistance in malaria parasites is associated with genetic mutations in target genes and can be monitored using molecular methods. CQ resistance is determined by the major point mutation at codon 76 of the P. falciparum CQ resistance transporter (pfcrt) gene [14], which is highly correlated with increased clinical CQ tolerance and treatment failure [14-16]. In addition, point mutations in P. falciparum multi-drug resistance gene 1 (pfmdr1) (e.g., N86Y, Y183F, S1034C, N1042D, and D1246Y) have been shown to modulate CQ resistance [17] and possibly lumefantrine resistance [18]. Resistance to antifolates is associated with point mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes [19]. Pyrimethamine (PY) resistance is conferred by the key mutation at codon 108 in the pfdhfr gene, while additional mutations at positions 51 and 59 increase the levels of resistance [20]. The 164L mutation common in Southeast Asia has been shown to confer PY resistance. Although this mutation is not common in Africa, a recent study by McCollin et al detected the 164L mutation in western Kenya [21]. Similarly, sulfadoxine (SD) resistance is conferred by a key mutation at codon 437 in the pfdhps gene and modulated by additional mutations at codons 436, 540, 581, and 613 [22]. Multiple mutations in these two genes result in antifolate treatment failure [23]. Molecular methods have been developed to detect point mutations in genes responsible for drug resistance [24-26], and have been widely used to monitor drug resistance in epidemiological surveys [16]. In this study, we have analyzed the mutations of antimalarial drug resistance genes for CQ (pfcrt and pfmdr1) and antifolates (pfdhps and pfdhfr) in samples from patients with acute malaria infections and school children with asymptomatic infections at one lowland and two highland locations in western Kenya.MethodsSample collectionSampling in two highland sites (Kisii, elevation ~1600 m above sea level; and Kakamega, elevation 1,480–1,560 m) and one neighboring lowland site (Kombewa, elevation ~1200 m) from June through August 2005 were conducted as a part of malaria surveillance activities. Average annual malaria prevalence among primary school children during the sampling period was 10.3%, 42.7% and 75% in Kisii, Kakamega and Kombewa, respectively. Malaria transmission intensity, measured by entomological inoculation rate (EIR), was 0.4, 16.6 and 31.1 infectious bites per person per year in Kisii, Kakamega and Kombewa, respectively [27]. At each site, 100 samples were collected from patients admitted with acute P. falciparum infection at a local hospital and 100 samples from primary school children (age 6–14 years old) with asymptomatic P. falciparum infections diagnosed by microscopy. Symptomatic malaria patients were treated by clinicians in the hospital with antimalarial drug, Coartem which achieved cure rates of up to 95%, even in areas of multi-drug resistance. Each sample consisted of ~200 μl of finger-prick blood spotted on filter papers. Filter papers were dried and stored at -20°C until parasite DNA extraction. The human subject protocol involved in this study has been approved by University of California, Irvine (UCI) and Kenya Medical Research Institute (KEMRI). Informed consent was provided by the parents/guardians of the children and assent from the children was obtained prior to the sample collection.Parasite DNA extraction and species identificationDNA was extracted from the blood filters using the Saponin/Chelex method [28]. Parasite DNA was extracted from one quarter of a blood spot of about 1 cm in diameter and dissolved in 200 μl of distilled water. Three microliters of the parasite DNA were used as the template for polymerase chain reaction (PCR). To avoid complications from mixed parasite species infections, a nested PCR method was used to verify P. falciparum infections and exclude the presence of other Plasmodium species in each sample [29]. Approximately 3–5% of samples containing mixed parasite species or other parasite species were identified in all the study sites. Samples containing only P. falciparum DNA were used for genotyping analysis.Molecular detection of mutations in drug targetsMutations in the pfcrt (K76T) and pfmdr1 genes were detected using a PCR-restriction fragment length polymorphism (RFLP) method. The fragment encompassing pfcrt codon 76 was amplified and digested with ApoI, which cleaves the wild type into 111 and 34 bp fragments [30]. Similarly, fragments containing codons 86, 184, 1034, 1042, and 1246 of the pfmdr1 gene were separately amplified by PCR and digested with respective restrictive enzymes as described previously [30-33]. Screening for mutations associated with antifolate resistance at codons 16, 50, 51, 59, 108, and 164 of the pfdhfr gene and codons 436, 437, 540, 581, and 613 of the pfdhps gene was performed by nested PCR and mutation-specific restriction enzyme digestions [34-36].Statistical analysisThe difference in frequencies of point mutations in the four aforementioned genes between highland and lowland parasite populations was determined by using the χ2 tests or Fisher's exact test for datasets with sample size less than 5. Yates' correction was applied for the chi-square value, resulting in corrected P values. Statistical significance was taken at the P = 0.05 level. Association between the different mutations was tested using Fisher's exact test.ResultsA total of 600 P. falciparum samples were analyzed for pfcrt, pfmdr1, pfdhfr, and pfdhps genes. Over 90% of samples were successfully amplified at the 17 test codons, and polymorphisms were detected at 10 of 17 codons screened (Table 1). Consistent with the hyperendemic settings in the study areas, we detected infections by mixed strains at seven of the 17 studied codons, which included codon 76 of pfcrt, codons 86, 184 and 1246 of pfmdr1, codon 59 of pfdhfr, and codons 437 and 540 of pfdhps (Table 1). The frequencies of mutant codons at the four genes varied slightly between the study sites, but the differences were not significant (Fig. 1). Mixed-genotype infections at pfdhfr codon 59, 437, and 540 of the antifolate target genes were low (<10%) in all samples. Similarly, mixed-genotype infections at codon 76 of the pfcrt gene were low (<15%). In contrast, mixed-genotype infections at codons 86, 184 and 1246 of the pfmdr1 gene were more frequent (18 – 45%) (Table 1).Table 1Frequencies of mutations in genes associated with resistance to chloroquine, sulfadoxine-pyrimethamine in P. falciparum parasites from symptomatic and asymptomatic volunteers in western Kenya.GeneMutationPolymorphismKombewaKakamegaKisiiS*A*SASApfcrtK76TMutant878186777570Mixed71569810Wild-type648141720Pfmdr1N86YMutant284128391023Mixed442245374018Wild-type283727245059Y184FMutant2415751126Mixed263023263319Wild-type505570695655D1246YMutant243740284224Mixed423036442129Wild-type343324283747pfdhfrN51IMutant989698989695Mixed000000Wild-type242245C59RMutant817588799086Mixed4102720Wild-type15151014814S108NMutant100100100100100100Mixed000000Wild-type000000pfdhpsS436FMutant424422Mixed000000Wild-type969896969898A437GMutant949592938893Mixed454720Wild-type2040107K540EMutant889592918893Mixed1054920Wild-type2040107* S – symptomatic; A – asymptomatic.Note: No mutations were detected at the following codons: pfdhfr (A16V, C50R and I164L); pfdhps (S436A, A581G and A613S); and pfmdr1 (S1034 and N1042D).Figure 1Frequency of mutations in Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) (A and B), multi-drug resistance gene (pfmdr1) (C and D) and quintuple mutations in dihydrofolate reductase (pfdhfr)/dihydropteroate synthetase (pfdhps) (E and F) genes in symptomatic and asymptomatic volunteers in highland (Kakamega and Kisii) and lowland (Kombewa) sites of western Kenya. The pfdhfr/pfdhps quintuple mutation refers to 51I/59R/108N/437G/540E. No mutations were detected at the following codons: pfdhfr (A16V, C50R, I164L and S436A), pfdhps (A581G and A613S), and pfmdr1 (S1034 and N1042D).Mutations in CQ resistance genesConsistent with the past extensive use of CQ in this area, the pfcrt 76T mutant allele was present in = 80% (including mixed infections with wild type and mutant alleles) of all samples from the three locations (Fig. 1A and 1B). In comparison, mutations at alleles 86Y, 184F and 1246Y of pfmdr1 gene were present at lower frequencies, ranging from 30 to 76% (including mixed infections) (Fig. 1C and 1D), whereas no mutation was found at codons 1034 and 1042, which was associated with CQ resistance in South America. The frequencies of mutations were generally not significantly different between the three study sites except pfcrt 76T and pfmdr1 86Y between Kombewa and Kisii (P < 0.05), and pfmdr1 184F between Kombewa and Kakamega (P < 0.01) (Fig. 1E and 1F). Consequently, the genotype 86Y/184Y/1246Y (wildtype at codon 184) was present at a significantly higher frequency in highland than in lowland parasites (P < 0.05), whereas the genotype 86Y/184F/1246Y (triple mutations) was significantly more frequent in lowland than in highland parasites (Table 2). Since certain pfmdr1 alleles were found associated with CQ resistance, we compared the association between pfmdr1 mutations and the major CQ-resistance determinant pfcrt 76T. When all parasite samples were taken into account, significant associations were found between the pfcrt 76T and two pfmdr1 alleles (184F and 1246Y) (P < 0.01). However, pfcrt 76T was not significantly associated with pfmdr1 86Y (P = 0.174), the mutation found associated with CQ-resistant field isolates in Asia and Africa.Table 2Percentage of haplotypes of P. falciparum parasites collected from symptomatic and asymptomatic volunteers in western Kenya.HaplotypesKombewaKakamegaKisisS*A*SASAPfmdr1  86Y/184Y/1246Y242644463836  86Y/184F/1246Y323424231613  86N/184F/1246D1215002527  86Y/184F/1246D18100510  86Y/184Y/1246D20161013  86N/184Y/1246D658101112  86N/184F/1246Y354385  86N/184Y/1246Y354304Pfdhfr/pfdhps Quintuple  51I/59R/108N/437G/540E878486838580 Quadruple  51I/59C/108N/437G/540E753844  51N/59R/108N/437G/540E004326  51I/59R/108N/437G/540K151212  51I/59R/108N/437A/540E110034 Triple  51I/59R/108N/437A/540K232254 Double  51I/59C/108N/437A/540K224200* S – symptomatic; A – asymptomatic; The mutation amino acids are underlined.Mutations in antifolate drug resistance genesDespite relatively recent deployment of antifolate drugs for controlling malaria in western Kenya, parasites with mutations in the drug target genes, pfdhfr and pfdhps, were highly prevalent. The mutation at codon 108 (allele 108N) of the pfdhfr gene, a major determinant of PY resistance, was ubiquitously present in all samples (Table 1). In addition, mutant alleles at codons 51 and 59 were also detected at high frequencies (85% – 98%). No mutation was detected at codons 16, 50, and 164 of pfdhfr. Most samples (90% – 100%) contained the key SD-resistance determinant mutation A437G and K540E in pfdhps. In contrast, the mutant 436F allele was rare (< 5%), and mutant 436A allele was not detected in any locations. Further, no mutation was detected at codons 581 and 613 of pfdhps.Increased numbers of mutations at the two antifolate target genes are associated with increased resistance to SD-PY. In the studied samples, single or double mutations were rarely encountered, whereas triple mutations in the pfdhfr gene were the most common. When the two genes were combined, quintuple mutations (51I/59R/108N of pfdhfr and 437G/540E of pfdhps) were found with a high prevalence (80–87%) at the three sites (Fig. 1E and 1F), followed by quadruple mutations (51I/59C/108N/437G/540E, 51N/59R/108N/437G/540E, 51I/59R/108N/437G/540K, 51I/59R/108N/437A/540E) (8% – 16%) (Table 2).Mutation frequency difference between asymptomatic and symptomatic samplesThe pfcrt K76T mutation frequencies were not significantly different in the asymptomatic and symptomatic samples in the lowland Kombewa site (96% vs. 94%; Fisher's exact test, P = 0.537) and two highland sites (86% vs. 92% in Kakamega, χ2 = 1.41, df = 1, P = 0.235; 80% vs. 83% in Kisii, χ2 0.03, df = 1, P = 0.863). Similarly, there was no significantly difference in mutation frequencies of pdmdr1 gene at test codons between asymptomatic and symptomatic samples in the lowland (Kombewa: χ2 = 1.44, df = 1, P = 0.230 at codon N86Y; χ2 = 0.33, df = 1, P = 0.566 at codon Y184F; χ2 = 0, df = 1, P = 1.000 at codon D1246Y) and two highland sites (Kakamega: χ2 = 0.07, df = 1, P = 0.791 at codon N86Y; χ2 = 0.01, df = 1, P = 0.920 at codon Y184F; χ2 = 0.26, df = 1, P = 0.610 at codon D1246Y. Kisii: χ2 = 0.87, df = 1, P = 0.351 at codon N86Y; χ2 = 0, df = 1, P = 1.000 at codon Y184F; χ2 = 1.40, df = 1, P = 0.237 at codon D1246Y). Similarly, for the pfdhfr/pfdhps quintuple mutation frequencies, no significantly difference was found in the asymptomatic and symptomatic samples in both lowland site (Kombewa: χ2 = 0.16, df = 1, P = 0.689) and highland sites (Kakamega: χ2 = 0.20, df = 1, P = 0.655. Kisii: χ2 = 0.32, df = 1, P = 0.572).DiscussionIn the present study, we analyzed the mutations of four known drug resistance genes in both highland and lowland parasite populations of western Kenya. We showed that the frequencies of key mutations in the pfcrt, pfmdr1, pfdhps, and pfdhfr genes that were implicated in resistance to CQ and SP were very high. We further demonstrated that there was no difference in the frequencies of key mutations between symptomatic and asymptomatic malaria volunteers. Our experiment design did not allow us to test clinical or parasitological efficacy in symptomatic infections after treatment; nor was the mutation prevalence/in vivo resistance before 1998 when the national policies of treating uncomplicated malaria were changed to SP was examined. Nevertheless, the big sample size in this study coupled to lack of differences in the frequency of resistant parasite genotypes in the two highland areas and the low land area clearly demonstrates a high frequency of drug resistant mutants circulating in the study areas.Resistance to CQ is largely determined by the K76T mutation in the pfcrt gene, and enhanced by mutations at other sites of this gene and mutations in the pfmdr1 gene. Since resistant phenotypes often have fitness costs [37], their prevalence is expected to decline after removal of the selective pressure. For example, the prevalence of mutant alleles of pfcrt 76T decreased from 64.5% in 2002 to 16% in 2004 and that of mutant pfmdr1 86Y alleles decreased from 46.6% to 2.7% two and half year after CQ withdrawal in coastal Tanzania [38]. Kublin et al. reported that the prevalence of the chloroquine-resistant pfcrt 76T genotype decreased from 85% in 1992 to 13% in 2000. In 2001, chloroquine cleared 100% of 63 asymptomatic P. falciparum infections, no isolates were resistant to chloroquine in vitro, and no infections with the chloroquine-resistant pfcrt 76T genotype were detected [39]. Similarly, Laufer et al. demonstrated that chloroquine was again an efficacious treatment for malaria, 12 years after it was withdrawn from use in Malawi [40]. Although Kenya had a similar drug policy, the pfcrt K76T mutation in western Kenya was still predominant. Consistent with the observation of high pfcrt K76T mutation frequency in the study sites, pfmdr1 mutations that enhance CQ resistance (e.g., 86Y) were also present at relatively high frequencies (37.2 – 45.2%) in western Kenya. This may be partly caused by wide availability and consumption of CQ after it was replaced as first-line antimalarial drug by antifolate drug (SP) in 1998 [12,13]. Our results indicated that parasite populations in western Kenya were still highly resistant to 4-aminoquinoline drugs.Several years after introduction of SP as the first-line antimalarial drug in Kenya, mutant genotypes have already become common. We found that mutations at codon 108 of the pfdhfr gene and codon 437 of the pfdhps gene, which are the major determinants for sulfadoxine and pyrimethamine resistance, respectively, were highly prevalent in our study sites. Furthermore, increased resistance to antifolate drugs was correlated with mutations at additional sites, which were also detected in our present study. In Kenya, Nzila et al. [41] reported that the pfdhfr triple mutant (codons 108, 51 and 59) was associated with seven-day treatment failure using Fansidar, and this association was strengthened by the presence of double mutations of the pfdhps gene at codons 437 and 540. Such \"quintuple\" mutants were also strongly correlated with Fansidar treatment failure in Malawi [26]. These quintuple mutants have already reached 80–87% prevalence in the parasite populations in western Kenya. This is consistent with the observation in another highland site in Chogoria, near Mount Kenya [42]. Since mutations at different sites of the pfdhfr and pfdhps genes might be a stepwise process, two mutations at codon 59 of pfdhfr and codon 540 of pfdhps can accurately predict the presence of this quintuple mutant [26]. The molecular method for monitoring antifolate resistance in P. falciparum can thus be simplified to detect the presence of these two mutations. This method has been shown to provide the best means of predicting clinical treatment outcomes in the patient population, which consists primarily of children from endemic areas of Africa [36]. In our study, either of these two mutations has exceeded 75% prevalence in the parasite populations, suggesting that at least 50% of the parasites carry these double mutations. Consistent with other genotype studies of pfdhfr from Kenyan isolates, we did not observe mutations at codon 164 [23,41,43], although McCollum et al. identified 164L mutation in several samples in western Kenya [21].The causes of the malaria epidemics in the East African highlands are complex and could be the result of interactions between environmental changes, vectors and parasites. Antimalarial drug resistance has been invoked as a major factor [9], however the molecular epidemiology of drug resistance in these highlands is unknown. Mbaisi et al. [43] reported a significantly lower prevalence of mutations at codon 437 of the pfdhps gene and codons 86 and 1246 of the pfmdr gene in lowland (Kisumu) samples than in highland (Kericho, Magadi, and Entosopia) samples in western Kenya, whereas this trend was the opposite for the mutation at codon 436 of the pfdhps gene. In contrast, our study did not detect significant differences in frequency of mutations at 14 codons in four drug resistant genes between samples from lowland and highland areas in western Kenya, although the prevalence of mutations at codons 86 and 1246 of the pfmdr gene was higher in samples from the lowlands (Kombewa) than in those from the highlands (Kakamega and Kisii). This result suggests either a similar drug selection pressure or significant gene flow between lowland and highland parasite populations aided by human travel [44], as detected by microsatellite and merozoite surface protein 1 and 2 (msp-1 and msp-2) [45].ConclusionTogether with the evidence that there was no significance difference in the frequencies of key resistance-conferring mutations in pfcrt, pfmdr1, pfdhps, and pfdhfr genes between symptomatic and asymptomatic malaria infections in western Kenya highland, high frequencies of these mutations in symptomatic and asymptomatic infections suggest that drug resistance of malaria parasites may be an important contributor to malaria-induced morbidity and mortality. However, the role of drug resistance as a driving force for malaria outbreaks in the highlands has not been established. If drug resistance were the main driving force, the lowland and highland sites should expect similar epidemic pattern of malaria incidence. Moreover, drug resistance level should increase over time until a new antimalarial drug is adopted. Thus, the number of malaria patients would be expected to increase gradually over time, but malaria incidence should not exhibit a large inter-annual variation. In contrast, dramatic fluctuations in malaria incidence in the African highlands were observed [9,46,47]. Significant association between climate variability and malaria incidence suggests that climate factors may play an important role in the East African highlands [47]. Further studies are required to examine the interactions between climate factors and drug resistance on malaria incidence in African highlands.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsDZ: Participated in the design of the study, conducted data collection, statistical analysis and drafting of the manuscript. YA: Conducted sample collection and helped with writing the manuscript. AG: Faciliated and conducted field sample collection. LC: Participated in the design of the study, interpretation of data and revising the manuscript. DM: Participated in experimental work and revising of the manuscript. GY: Conceived the study, participated in collection of samples, and participated in manuscript preparation. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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+    "id": "PMC2533350",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533350\nAUTHORS: Susanna Y Huh, Ruth Andrew, Janet W Rich-Edwards, Ken P Kleinman, Jonathan R Seckl, Matthew W Gillman\n\nABSTRACT:\nBackgroundAnimal data show that decreased activity of placental 11-beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which potently inactivates glucocorticoids (e.g. cortisol) to inert forms (cortisone), allows increased access of maternal glucocorticoids to the fetus and 'programs' hypertension. Data in humans are limited. We examined in humans the association between venous umbilical cord blood glucocorticoids, a potential marker for placental 11β-HSD2 enzyme activity, and blood pressure at age 3 years.MethodsAmong 286 newborns in Project Viva, a prospective pre-birth cohort study based in eastern Massachusetts, we measured cortisol (F) and cortisone (E) in venous cord blood and used the ratio of F/E as a marker for placental 11β-HSD2 activity. We measured blood pressure (BP) when the offspring reached age 3 years. Using mixed effects regression models to control for BP measurement conditions, maternal and child characteristics, we examined the association between the F/E ratio and child BP.ResultsAt age 3 years, each unit increase in the F/E ratio was associated with a 1.6 mm Hg increase in systolic BP (95% CI 0.0 to 3.1). The F/E ratio was not associated with diastolic blood pressure or birth weight for gestational age z-score.ConclusionA higher F/E ratio in umbilical venous cord blood, likely reflecting reduced placental 11β-HSD2 activity, was associated with higher systolic blood pressure at age 3 years. Our data suggest that increased fetal exposure to active maternal glucocorticoids may program later systolic blood pressure.\n\nBODY:\nBackgroundMany observational studies have demonstrated an inverse association between birth weight and blood pressure (BP), raising the intriguing possibility that lifelong hypertension might be programmed in utero [1]. The mechanisms underlying this association remain unclear.One plausible hypothesis, based on animal data, proposes that a low protein diet during pregnancy leads to increased fetal glucocorticoid exposure, permanently programming both lower birth weight and elevated BP in offspring [2]. Studies in humans have not found a consistent association between maternal protein intake and offspring blood pressure [3-6], but few studies have examined whether fetal glucocorticoid exposure might program offspring BP.Experiments in rats have shown that maternal treatment during pregnancy with the synthetic glucocorticoid dexamethasone, which freely crosses the placenta, is associated with lower birth weight and raised BP in the offspring [7]. Protection from exposure to maternal physiological glucocorticoids is mediated by a placental enzyme, 11-beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which converts active glucocorticoid (corticosterone in rats, cortisol in humans) into inert 11-keto forms [7]. The 'placental barrier' function of 11β-HSD2 is important because circulating maternal cortisol freely crosses the placenta.A relative deficiency of placental 11β-HSD2 might allow increased access of maternal glucocorticoids to the fetus, retarding growth and programming responses leading to later hypertension, as suggested by studies in rats and mice [7-9]. Humans who are homozygous for the gene encoding 11β-HSD2, resulting in little or no enzyme activity, show much lower birth weight than their unaffected (mainly heterozygous) siblings [10]. Limited data suggest that heterozygous carriers may have a milder phenotype indistinguishable from essential hypertension, though dissecting placental from offspring renal effects is problematic [11].We hypothesized that a higher ratio of cortisol to cortisone in umbilical venous cord blood, reflecting decreased 11β-HSD2 activity, would be associated with lower birth weight and raised blood pressure in childhood. We examined this hypothesis within Project Viva, a longitudinal cohort of pregnant women and their offspring.MethodsSubjectsWe recruited women with a singleton pregnancy from April 1999 to July 2002 at eight obstetric offices in the Boston, Massachusetts, USA area [12]. Women were study-eligible if they entered prenatal care before 22 weeks of gestation, planned ongoing obstetric care at an enrollment site, and were able to answer questionnaires in English. We collected sociodemographic and medical data through in-person interviews, self-administered questionnaires and hospital and ambulatory medical records. Human Subjects Committees of Harvard Pilgrim Health Care, Brigham and Women's Hospital, and Beth Israel Deaconess Medical Center approved study protocols. All participants gave informed consent [12].We previously described study population, enrollment and follow up procedures [12]. Of 657 infants with cord blood stored at 4°C for less than 30 hours, 362 infants were eligible for this analysis (mothers consented to enroll their infants into the study and completed a research visit including blood pressure at age 6 months). Of the 362 eligible infants, 286 (79%) returned for a 3-year blood pressure measurement. Therefore, we based our analyses on 286 children with 3-year blood pressure.Umbilical cord blood glucocorticoidsAt the time of delivery, using a needle and syringe technique, a midwife or obstetrician collected cord venous blood. Because research technicians were unable to be present at every delivery, delivery room staff refrigerated the specimen immediately after collection for a period of up to 30 hours (median: 13 hours). A research technician then separated the specimen into aliquots of serum, red blood cells, and white blood cells for storage at -70°C until analysis. We performed a small validation study to ensure that refrigeration of samples for less than 30 hours did not lead to degradation of glucocorticoid levels. We collected blood samples from 11 pregnant women and used three different methods of storage and processing. In method A (the gold standard), blood was drawn into room temperature heparinized glass tubes, spun at room temperature immediately, and the plasma was frozen at -20°C for 24 hours before being transported to a storage site and stored at -80°C for 1–6 months. In method B, blood was treated similarly except that after being drawn it was refrigerated at 4°C for 4 hours, and in method C for 24 hours. We found that within-person cortisol levels did not significantly differ by cortisol processing protocol. Comparing methods A and B, the mean within-person difference in cortisol was 22.1 nmol/liter (95%CI -129.7 to 173.8, p = 0.76). Comparing methods A and C, the mean within-person difference in cortisol was 60.7 nmol/liter (95% CI -80.0 to 204.2, p = 0.36).Cortisol and cortisone were quantified in duplicate in venous cord plasma by validated direct radioimmunoassay techniques [13,14]. Concentrations of cortisol and cortisone were determined using a radioimmunoassay kit for cortisol (MP Biomedicals, UK) and cortisone (Immunovation Ltd, Southampton, UK) [15]. The intra-assay coefficients of variation were 5.6% and 5.2% for cortisol and cortisone respectively.We used the mean of duplicate cortisol and cortisone assays to calculate our primary exposure, the ratio of cortisol/cortisone in umbilical venous blood, which correlates well (r = 0.5) with direct measures of 11β-HSD2 activity using placental tissue homogenates [16].Blood pressure and anthropometric measurementsAt the 3-year visit, trained research assistants measured each child's blood pressure (BP) up to five times, at 1-minute intervals, using a Dinamap Pro 100 or Pro 200 (Critikon Inc., Tampa, FL, USA) automated blood pressure monitor. We recorded the conditions of measurement as previously described [3], including room temperature, activity state of the child (crying, quiet awake, active awake), cuff size (infant, child, small adult), appendage used for BP measurement (left arm, right arm, calf), and child position (semi-reclining, seated, standing). The majority of children, 255 out of 286 (89%), had five blood pressure measurements (mean 4.8), and 87% of measurements were taken when the child was in a quiet, awake state. We defined our primary endpoint to be child systolic BP, because of its validity of measurement with an automated device [17]. We examined diastolic BP as a secondary outcome.We measured child weight with a digital scale, and height using a stadiometer (Shorr Productions, Olney, MD, USA). We calculated a continuous measure of sex-specific birth weight for gestational age z-score (fetal growth) based on published US reference standards [18]. We computed gestational age at birth as the number of days between the first day of last menstrual period and the delivery date and confirmed by ultrasound fetal measurements at 16–20 weeks. For date discrepancies of more than 10 days, we used the ultrasound-based gestational age.Statistical methodsTo assess multivariable associations between cord glucocorticoids and offspring BP, we used mixed effect regression models, incorporating each of the up to five BP measurements per child as repeated outcome measures [19]. In contrast to standard ordinary least squares analysis, mixed effect models weight subjects based on the number of measurements and their variability, and thus result in appropriate standard errors. In our baseline crude model, we included only BP measurement conditions to reduce measurement error in child BP. Crude models stratified by gender showed similar associations among males and females; therefore we combined males and females in our models, adjusting for gender. In our multivariable model, we added gestational age at birth, child age, gender, attained length and weight, and maternal race and income [18]. Potential confounders that did not change our effect estimates included maternal age, pre-pregnancy body mass index, maternal height, gravidity, pregnancy weight gain, third-trimester BP, induced delivery, Caesarean section, Apgar score, smoking status prior to pregnancy, education level, and marital status. Therefore, we excluded these variables from our final models. We conducted all data analyses using SAS version 9.1 (SAS Institute Inc., Cary, NC, USA).ResultsParticipant characteristics are shown in Table 1. Most mothers had a relatively high level of income and education. Mean venous cord cortisol was 343.9 nmol/liter (74.5–1042.9, 1st to 99th percentile). Mean venous cord cortisone was 242.7 nmol/liter (106.3–484.0, 1st to 99th percentile). Mean F/E ratio was 1.4 (0.5–3.8, 1st to 99th percentile). The Pearson correlation of the F/E ratio with cortisol was 0.8, and with cortisone was -0.1. The correlation of F/E ratio with birth weight for gestational age z-score was 0.02, and with gestational age at birth was 0.1. Mean systolic blood pressure was 93.3 mm Hg at age 3 years. Mean diastolic blood pressure was 58.9 mm Hg.Table 1Cord blood glucocorticoids and other characteristics of 286 participants with 3-year blood pressure.nMean (SD) or %Maternal characteristics at enrollment Age (years)28632.2 (4.9) Pre-pregnancy BMI (kg/m2)28424.6 (5.1) Race/ethnicity (%)  White20471%  Black3111%  Hispanic207%  Asian135%  Other186% Marital status (%)  Married23883%  Partner269%  Other228% Education (%)  High school or less176%  Less than 4 years of college7326%  4 years of college10035%  Graduate degree9634% Annual household income (%)  <$20 00031%  $20 000–$40 0003010%  $40 000–$70 0006523%  >$70 00017361%  Missing155% Smoked 3 months prior to pregnancy (%)238% Pregnancy weight gain (kg)28315.6 (5.5) 3rd trimester systolic BP (mm Hg)285110.9 (8.3) Spontaneous delivery (vs induced)18665% Caesarean section (vs vaginal delivery)4716%Child characteristics Male (%)14651% Birth weight (g)2863545 (501) Birth weight for gestational age z-value (units)2860.2 (0.9) Gestational age (weeks)28639.7 (1.3) Cortisol (nmol/liter)286343.9 (209.3) Cortisone (nmol/liter)286242.7 (81.3) Cortisol/cortisone ratio (units)2861.4 (0.6) Systolic BP at age 3 years (mm Hg)28693.3 (10.2) Diastolic BP at age 3 years (mm Hg)28658.9 (8.0) Weight at age 3 years (kg)28615.8 (2.6) Height at age 3 years (cm)28697.6 (4.6)BP, blood pressure.Compared with the 657 mother-offspring pairs with cord blood, mothers included in our analyses had a slightly higher level of education (69% vs 62% completed a college or graduate degree) and had a higher household income (60% vs 54% reported income of more than $70,000 per year). Mean birth weight was similar among infants included in our analyses compared with all eligible infants (3545 g vs 3529 g). Child systolic and diastolic blood pressure, length and weight were similar among included and eligible children.Bivariate analyses showing systolic BP as a function of F/E ratio are shown in Figure 1. In multivariable analyses, each one unit increment in F/E ratio was associated with a 1.6 mm Hg (95% CI 0.0 to 3.1, p = 0.05) increment in systolic BP at age 3 years (Table 2). The direction of this effect estimate at age 3 years was consistent with our hypothesis that higher F/E ratio, reflecting lower 11β-HSD2 activity, would be associated with higher blood pressure. The effect estimate for diastolic BP at 3 years was 0.8 mm Hg (95% CI -0.4 to 2.0), adjusted for other covariates.Table 2Multivariable mixed effect models showing change in 3-year child BP per unit increment in venous cord F/E ratio.Change in child BP (mm Hg) per one unit increment in F/E ratio (95% CI)Models*Systolic BPDiastolic BPModel 1: F/E ratio (unit increment)1.2 (-0.4 to 2.7)p = 0.140.7 (-0.5, 1.8)p = 0.27Model 2: Model 1 + infant sex, gestational age at birth, age, 3 year weight, length, maternal race/ethnicity, income1.6 (0.0 to 3.1)p = 0.050.8 (-0.4, 2.0)p = 0.20Data from 286 mother-offspring pairs within Project Viva.*All models were adjusted for blood pressure measurement order, cuff size, appendage, position, state, and machine model. BP, blood pressure; F/E, cortisol/cortisone ratio in venous cord blood.Figure 1Systolic blood pressure (BP) at age 3 years as a function of cortisol/cortisone (F/E) ratio. Bivariable regression line (solid line), unadjusted for covariates, and its 95% confidence limits (dotted lines) are shown.In unadjusted analyses, the F/E ratio was not associated with birth weight; the effect estimate for birth weight was 47.1 g per one unit increment in the F/E ratio estimating placental 11β-HSD2 activity (95% CI -42.9 to 137.1). After adjustment for gestational age and sex, there was no association between F/E ratio and birth weight (-0.2 g for each one unit increment in F/E ratio, 95% CI -76.1 to 75.7). After additional adjustment for maternal age, BMI, race, income, and smoking status, the effect estimate was -12.1 g per unit increment in F/E ratio (95% CI -87.0 to 62.8). In unadjusted analyses, the effect estimate for the association between F/E ratio and gestational age at birth was 0.2 weeks per one unit increment in the F/E ratio (95% CI -0.0 to 0.5, p = 0.05). Additional adjustment for maternal age, BMI, race, income, and smoking status did not materially change the effect estimate (0.2 weeks, 95% CI -0.05 to 0.5, p = 0.11).DiscussionOur study is the first in humans to examine the relationship between umbilical cord concentrations of glucocorticoids and offspring blood pressure. We found that a one unit increment in F/E ratio, a proxy for reduced placental 11β-HSD2 activity, was associated with a 1.6 mm Hg higher systolic BP at 3 years of age. Although this effect estimate may appear to be small, a shift of this magnitude in the mean population blood pressure could result in a clinically meaningful difference in the prevalence of hypertension. A meta-analysis of 1 million adults estimated that a 2 mm Hg reduction in systolic blood pressure could result in a 10% decrease in mortality from stroke, and a 7% decrease in mortality from ischemic heart disease [20]. We did not find an association between F/E ratio and fetal growth, suggesting that lower activity levels of 11β-HSD2 might program higher offspring blood pressure without restricting overall fetal growth.Our finding is consistent with data in animals showing that decreased placental 11β-HSD2 activity programs higher offspring blood pressure. Rats and sheep treated during pregnancy with the glucocorticoid dexamethasone, a relatively poor substrate for 11β-HSD2, have offspring with reduced birth weight and elevated blood pressure in offspring [7]. Pregnant rats given an 11β-HSD2 inhibitor (carbenoxolone) have offspring with lower birth weight and raised blood pressure [8]. In addition, pregnant rats fed a low protein diet have been shown to have both decreased placental 11β-HSD2 activity and higher offspring BP [2]. Within Project Viva, we did not find a relationship either between protein intake and infant BP [3] or between protein intake and F/E ratio (data not shown).No human studies have directly examined the relationship of placental 11β-HSD2 activity or cord glucocorticoid levels with later blood pressure. A few studies in humans have examined the effect of prenatal exogenous steroid administration on later blood pressure. In one observational study, preterm infants exposed to antenatal betamethasone had higher systolic BP and diastolic BP in adolescence than unexposed infants [21]. However, follow-up data from two randomized controlled trials using two doses of prenatal betamethasone found that prenatal betamethasone use either did not affect offspring blood pressure [22,23], or was associated with a lower systolic blood pressure in offspring [24]. It is possible that the effects of 11β-HSD2 activity could differ among preterm and term infants. Several [16,25,26], but not all [27-29], studies suggest that human placental 11β-HSD2 expression or activity rises with increasing gestational age until term; our data suggested a direct correlation between F/E ratio and gestational age at birth, although the effect estimates did not reach statistical significance. Preterm infants may also be predisposed to higher blood pressures in later life for other reasons [30]. Our study excluded infants with a gestational age of less than 33 weeks and may therefore be more generalizable than prior studies focusing on preterm infants.In animals, lower placental 11β-HSD2 activity is associated with lower birth weight [8]. In humans, similar associations have been shown in preterm infants [16,31]. Among term infants, two studies did not find an association between 11β-HSD2 activity and birth weight [32,33]. One study found that among asthmatic pregnant women with a female fetus, lower birth weight was associated with reduced placental 11β-HSD2 activity [34]; this association was not present among women pregnant with a male fetus [34], suggesting that there may be sex-specific differences in regulation of 11β-HSD2 activity. Our data (mean gestational age 39.7 weeks) showed little association between the F/E ratio and fetal growth, suggesting that a relative deficiency in placental 11β-HSD2 activity might program higher offspring blood pressure without affecting birth weight. Stratification by child gender made no difference to our analyses. Because birth weight has many determinants, it is not surprising that physiologic programming of blood pressure might occur without affecting birth weight [35].Our results must be interpreted with caution, because we used F/E ratio in venous cord blood as a proxy for placental 11β-HSD2 activity rather than directly measuring enzyme activity. Glucocorticoids in the human fetal circulation may originate from the fetal adrenal gland, or from maternal cortisol, which freely crosses the placenta. Data show that 75–100% of cord blood cortisone originates from placental metabolism of maternal cortisol [36]. Cortisol levels in venous umbilical cord blood may be affected by increased production of cortisol by fetal adrenal glands at term, as well as maternal or fetal stress and other factors [37]. Adjustment for mode of delivery and induction of labor made no difference to our estimates; other variables relating to labor and delivery, such as duration of labor, or the use of instrumentation were not available for these analyses. Prior studies have not found an effect of labor on 11β-HSD2 mRNA or activity levels [29,38].Among healthy term infants, the ratio of cortisol/cortisone in umbilical venous cord blood appears to correlate well with direct measures of placental 11β-HSD2 activity using placental tissue homogenates [16]. A study that measured placental cortisol metabolism using a similar venous cord ratio, E/(E+F), was not improved by creation of an 11β-HSD2 activity index incorporating arterial cord glucocorticoid measurements [39], which presumably reflect the fetal adrenal contribution (Pearson correlation between E/(E+F) and F/E was 0.9 using our data). The cord F/E ratio could also be affected by the activity of either placental 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1) or fetal 11β-HSD2. 11β-HSD1, an enzyme isoform that can convert cortisone to cortisol, has been detected in human perfused placenta [40], and placental 11β-HSD1 activity levels may increase during gestation [41]. However, studies suggest that type 2 enzymatic activity predominates during pregnancy [27,42], with one study reporting no detectable 11β-HSD1 activity [43]. Several fetal tissues express 11β-HSD2 until mid-gestation [44]. Dy et al. recently reported that among term infants born with intrauterine growth restriction, the ratio of cortisone to cortisol in the umbilical artery was lower than in the umbilical vein, suggesting either attenuated fetal 11β-HSD2 activity, or reduced fetal glucocorticoid clearance [45]. Term infants with a birth weight appropriate for gestational age showed no difference in cortisone to cortisol ratio between the umbilical artery and vein [45]. Since most 11β-HSD2 expression is lost from the fetus by term (at least in rodents and probably in humans) we think this is unlikely to have contributed to levels of cortisol and cortisone in cord blood, emphasizing the placental contribution.Among infants born preterm or small for gestational age, the F/E ratio may not be a valid proxy for placental 11β-HSD2 activity. A study of preterm infants born at less than 32 weeks gestational age was unable to detect a correlation between venous cord blood glucocorticoid concentrations and placental 11β-HSD2 activity [31]. A recent study reported that compared with term infants with a birth weight appropriate for gestational age, infants with intrauterine growth restriction had reduced placental homogenate 11β-HSD2 activity, but similar umbilical cord venous cortisone to cortisol ratio [45]. The reasons underlying the lack of correlation between the venous cord F/E ratio and placental 11β-HSD2 activity among preterm and small for gestational age infants are unclear, but might be explained by a greater contribution of placental 11β-HSD1 activity to circulating glucocorticoid levels, or decreased glucocorticoid clearance by the placenta or fetal tissues [45]. Our study included only infants born later than 33 weeks gestation (mean 39.7 weeks), and the majority of our participants were healthy term infants with a birth weight appropriate for gestational age (mean birth weight 3545 g), supporting the use of F/E as a valid proxy for placental 11β-HSD2 activity in our study.Strengths of our study include our adjustment for multiple relevant confounders and careful blood pressure measurements. Our study has several limitations. One limitation is that we measured glucocorticoids only at a single time point (delivery), and thus were unable to measure the potential impact of presumed fetal glucocorticoid exposure at earlier time windows during pregnancy. By definition, use of a proxy for enzymatic activity results in some misclassification of the exposure. A non-differential misclassification should have biased our findings toward the null; therefore, our data may actually underestimate the association between fetal glucocorticoid exposure and child BP. Our study did have some loss to follow-up, and therefore may be subject to selection bias. However, child BP among participants excluded from our analyses was similar to those included in our analyses. It seems unlikely that placental 11β-HSD2 activity would systematically differ in participants excluded from our analyses, although we cannot exclude this possibility.ConclusionIn conclusion, our data show for the first time that a higher F/E ratio in venous cord blood is associated with higher offspring systolic blood pressure in humans. The higher F/E ratio may reflect reduced placental 11β-HSD2 activity, resulting in increased fetal glucocorticoid exposure and higher offspring systolic blood pressure. Our study goes beyond previous data focusing on the birth weight-blood pressure relationship to explore a potential prenatal mechanism for the programming of later blood pressure, but our findings are limited by the use of an indirect measure of enzyme activity. Additional studies using direct measures of enzymatic activity are needed to definitively determine whether placental 11β-HSD2 activity does program blood pressure in childhood.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsSYH designed research question, led data analysis, and drafted the manuscript. RA directed glucocorticoid assays, assisted in study design and data analysis, and contributed to the manuscript. KPK designed the statistical analysis and contributed to the manuscript. JWR–E obtained funding, helped direct study operations, and contributed to the manuscript. JRS participated in the study design and contributed to the manuscript. MWG participated in the study design, obtained funding, directed study operations, and contributed to the manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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+    "id": "PMC2533352",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533352\nAUTHORS: David McCoy, Barbara McPake, Victor Mwapasa\n\nABSTRACT:\nTwo crises dominate the health sectors of sub-Saharan African countries: those of human resources and of HIV. Nevertheless, there is considerable variation in the extent to which these two phenomena affect sub-Saharan countries, with a few facing extreme levels of both: Lesotho, Zimbabwe, Zambia, Mozambique, the Central African Republic and Malawi.This paper reviews the continent-wide situation with respect to this double burden before considering the case of Malawi in more detail. In Malawi, there has been significant concurrent investment in both an Emergency Human Resource Programme and an antiretroviral therapy programme which was treating 60,000 people by the end of 2006. Both areas of synergy and conflict have arisen, as the two programmes have been implemented. These highlight important issues for programme planners and managers to address and emphasize that planning for the scale-up of antiretroviral therapy while simultaneously strengthening health systems and the human resource situation requires prioritization among compelling cases for support, and time (not just resources).\n\nBODY:\nBackgroundTwo crises dominate the health sectors of sub-Saharan countries: those of human resources and of HIV. In principle, both these crises magnify each other. HIV places a significant additional load on the health workforce and contributes to attrition from it through illness, caring for family members who have developed AIDS and death. And the impact of the HIV crisis is accentuated because health workers are unavailable to implement anti-HIV interventions.A particular source of recent concern has been the impact on workforce distribution of increased levels of support for HIV/AIDS programmes and especially treatment. This paper seeks to explore this interaction in more detail. It reviews the continent-wide distribution of the two phenomena and initial evidence of the impact of expanded treatment programmes, before looking in depth at the case of Malawi, a country with one of the lowest densities of human resources for health and one of the highest prevalence rates of HIV.MethodsThis paper is based on data derived from published literature; the global atlas of the health workforce, a database compiled by the World Health Organisation (WHO); and grey literature, particularly concerning Malawi. In addition, one of the authors (DM) was part of a nine member external team established by the Government of Malawi and the UK Department for International Development to evaluate the country's antiretroviral therapy (ART) programme in September 2006. During the evaluation a number of health facilities were visited and informal interviews and discussions with service providers, managers and policy makers were conducted.FindingsThe twin human resource and HIV burdenThe 2006 World Health Report (WHR) defined health workers as 'the people whose job it is to protect and improve the health of their communities' [1]. While recognising the important role of unpaid carers such as mothers and voluntary health workers, its analysis is restricted to people engaged in paid activities. Among those, two categories are identified: 'health service providers' who deliver services; and 'health management and support workers' who are not engaged in any direct provision of services. Table 1, reproduced from the WHR, summarises data on the availability of health workers by region and by the categories mentioned above. It suggests that regions with more health workers have proportionately more managerial and support workers. However, better data are required before any conclusions can be made about the number and relative availability of 'health management and support workers'.Table 1Global health workforce, by densityWHO RegionTotal health workforceHealth service providersHealth management and support workersNumberDensity (per 1000 population)Number% of total health workforceNumber% of total health workforceAfrica1 640 0002.31 360 00083280 00017Eastern Mediterranean2 100 0004.01 580 00075520 00025South-East Asia7 040 0004.34 730 000672 300 00033Western Pacific10 070 0005.87 810 000782 260 00023Europe16 630 00018.911 540 000695 090 00031Americas21 740 00024.812 460 000579 280 00043World59 220 0009.339 470 0006719 750 00033Note: All data for latest available year. For countries where data on the number of health management and support workers were not available, estimates have been made based on regional averages for countries with complete data.Data sources: World Health Organization. Global Atlas of the Health Workforce The WHR also identified those countries with a 'critical shortage' of health workers (see Figure 1). Critical shortage was defined as having less than 2.28 doctors, nurses and midwives per 1000 population, a threshold derived from an analysis of workforce density associated with key public health outcomes by the Joint Learning Initiative [2] (see Table 1).Figure 1Countries with a critical shortage of health service providers (doctors, nurses and midwives). (Source: World Health Report (2006), Working Together for Health, Geneva: WHO [1]).The WHR suggest that there are critical shortages of health workers in many countries. The absolute shortage is greatest in Asia, where there is a shortfall of 1.16 million doctors, nurses and midwives and perhaps 2.1 million of all types of health workers, dominated by the shortages in Bangladesh, India and Indonesia. The relative shortage is greatest in sub-Saharan Africa where an increase of 139% is required [1]. The countries with the lowest ratios of health workers per 1000 population are mainly in sub-Saharan Africa but some others such as Indonesia and Papua New Guinea also have densities below one half of the proposed critical shortage threshold.The focus on doctors, nurses and midwives reflects the greater reliability of estimated numbers of these cadres. In practice, the contribution of other cadres such as pharmacists, laboratory technicians and 'non-physician clinicians', is just as critical. Indeed, 'non-physician clinicians' (often known as 'clinical officers' or 'medical assistants') have been trained in some countries to compensate for the lack of doctors and are active in 25 of 47 sub-Saharan African countries included in a recent study [3]. In nine countries there are more non-physician clinicians than physicians and they are reported to play prominent roles in primary health care and HIV/AIDS treatment in five of the worst affected sub-Saharan countries. However, in no country do they add more than 0.2 to the health worker per thousand population ratio, so they do not significantly alter the relative position of different countries from WHO's analyses.Further analysis of data from WHO's global atlas of the health workforce identifies the countries in Table 2 as having ratios of doctors, nurses and midwives lower than 0.5 per 1000 population. All of these are in sub-Saharan Africa. Malawi has a slightly higher overall health worker density than these countries at 0.61 per thousand population, but a physician density level as low as the least well served of these countries (Niger) at 0.02 per thousand population [3] (see Table 2).Table 2Countries in the deepest human resource crisis according to their numbers of doctors, nurses and midwives: ratios per thousand population.PhysiciansNursesMidwivesTOTALBurundi0.030.1900.22Ethiopia0.030.20.020.25Niger0.020.20.030.25Chad0.040.240.040.32Liberia0.030.170.130.33Mozambique0.030.210.120.36Senegal0.060.250.070.38United Republic of Tanzania0.020.30.070.39Togo0.040.330.050.42Rwanda0.050.420.010.48Central African Republic0.080.230.180.49Source: Author's analysis of HRH global atlas, latest year available The focus on nurses, doctors and midwives also runs the risk of neglecting the importance of 'health management and support workers'. Clinical workers require management and administrative systems to work if they are to be effective. And ART programmes require, in particular, effective drug procurement and supply systems, laboratory support and information management.When countries with low HR levels are assessed in terms of HIV prevalence, all the non-African countries with a critical human resource shortage are found to have relatively low adult HIV prevalence rates. According to UNAIDS [4], adult HIV prevalence ranges from less than 0.1 to 1.6% in these countries except Haiti, where prevalence is 3.8%. The twin burden of HRH crisis and HIV/AIDS crisis is therefore an African phenomenon. Figure 2 plots total numbers of doctors, nurses and midwives against adult HIV prevalence across all African countries for which both statistics are available. It identifies 6 countries with an HRH crisis as defined by WHO and with adult HIV prevalence rates greater than 10%. These are Lesotho, Zimbabwe, Zambia, Mozambique, the Central African Republic and Malawi (see Figure 2).Figure 2Total numbers of doctors, nurses and midwives against adult HIV prevalence across African countries for which both statistics are available. (Source: Authors' analysis based on HRH global atlas and UNAIDS data).Hirschhorn et al. [5] estimated that the additional health workforce required to deliver ART to 1000 patients amounted to 1–2 physicians, 2–7 nurses, <1 to 3 pharmacy staff and an unquantified number of counsellors and treatment supporters. On this basis, Mozambique, which needs to provide ART to about 200,000 patients, would require 200–400 doctors from its' total stock of 514, and 400–1400 nurses from its total stock of 3947.Other estimates of the workforce required to scale up ART suggest even more stark results. Smith [6] calculated that seven out of fourteen countries included in his study would be unable to meet needs even if they used 100% of their current workforce. Figure 3 shows Smith's estimates of human resource requirements for full coverage of population with antiretroviral therapy. Only two of the 'twin burden' countries are considered in Smith's analysis – Mozambique and Zambia (Malawi was not included). Both are among the three countries whose current medical personnel situation appears least adequate for antiretroviral therapy expansion according to Smith. The third is Rwanda, one of the most human resource constrained countries (see Table 2), but with a relatively low estimated HIV prevalence rate. This estimate has recently fallen from a reported rate of 8.9% to 3.1% following the expansion of sentinel HIV surveillance to rural sites [7]; it is possible, but not clear, if Smith used the higher rate in his calculation (see Figure 3).Figure 3Percentage of existing doctor workforce required for full coverage in 10 years. (Source: Smith 2005 [6]).In part, these stark estimates reflect the clinical complexity and chronic nature of treating patients with AIDS. Even in the absence of antiretroviral therapy, HIV increases the needs for skilled human intervention in the health system, particularly due to the incidence of opportunistic infections. For example, one study in Rwanda estimated that 60% of hospital beds were occupied by AIDS patients being treated for opportunistic infections [8].A comprehensive HIV/AIDS programme also includes a range of interventions unrelated to the treatment of people with AIDS such as HIV prevention strategies, including the comprehensive management of patients with other sexually transmitted infections, voluntary counselling and testing (VCT) services and the prevention of vertical transmission. All these interventions also require skilled health workers.The HR requirements of ART programmes therefore have to be met within a severely limited pool of human resources. It is therefore unsurprising that the volume of additional funding and energy directed at HIV/AIDS programmes should threaten less well supported activities. Furthermore, the delivery of HIV/AIDS interventions through non-government organisations (NGOs) and private providers that are able to offer better pay and working conditions to health workers can lead to attrition from the public sector and other areas of health care [9,10].A case study of MalawiBackgroundWith an estimated GDP per head of US$646 in 2004, Malawi is one of the poorest countries in Africa [11]. Over half the 12 million population is food insecure and 65.3% were unable to meet their daily consumption needs in 1998 [12]. Life expectancy at birth is 39.8 years. HIV prevalence in Malawi was 14.1% (CI: 6.9 – 21.4) in 2005 [13]. The country is heavily dependent on aid which contributed 31.2% of Gross National Income in 2003, a higher proportion than most other countries in sub-Saharan Africa [14].Malawi's health care indicators are poor [15-17]:• Only 10% of health facilities were able in 2002 to deliver a basic minimum standard of care, with many being in poor condition, lacking an operational water source, electricity or a working telecommunications system.• Full immunisation coverage has fallen from a rate of 81.8% in 1990 to 64.4% in 2004.• The maternal mortality ratio is one of the highest in the world, standing at 984 per 100 000 live births in 2004.• Only 46% of the population live within 5 km of a formal health facility and only 20% live within 25 km of a hospital• 73% of households lacked an insecticide-treated bednet in 2004. According to one survey, more than 50% of malaria cases do not get treatment at health facilities.There are however some notable achievements. Neonatal tetanus and polio have been eliminated through immunisation programmes and TB cure rates are over 70% [18]. And, as discussed later, there has been a great increase in the number of people living with AIDS receiving anti-retroviral therapy.Malawi's health system is severely under-financed. In 2001, total health expenditure was US$ 12.4 per person [19]. At that time, the cost of delivering an 'essential health package' (EHP) of eleven cost-effective health services was estimated at $17.53 per capita, nearly 50% more than existing total health spending [20]. Furthermore, the cost estimate of this EHP was based on only 67% coverage for some services and did not include the costs of central level management and supervision, central hospital activity, or the provision of antiretroviral therapy.According to WHO's National Health Accounts database, per capita total health expenditure in 2005 had risen to US$ 23. The government accounted for 24.3% of total health spending; donors/external funding for 51.5%; and private expenditure for 24.2% [21]. The organization of health care finances in Malawi has improved since 2005 as a consequence of a Sector Wide Approach (SWAp) which several donors, particularly DFID (UK), have agreed to support. Under the SWAp, a six-year programme of work was established, with the delivery of the EHP being at the core. However, not all external funding is channelled through the SWAp. USAID and PEPFAR are notable bilateral donors operating outside the SWAp framework.In line with the focused international attention on HIV/AIDS, Malawi established a separate National AIDS Commission (NAC) to manage the significant amount of dedicated HIV/AIDS funding (including grants from the Global Fund) and to provide oversight over the country's HIV/AIDS plan. When first established, tension existed between the NAC and the Ministry of Health, partly because the NAC employed staff at higher salaries than the Ministry and because of the Ministry's loss of direct control over HIV/AIDS funding. According to a draft copy of Malawi's 2004/05 National Health Accounts, the Ministry of Health's share of public finance has decreased between 2002/03 and 2004/05 while that of the NAC increased (see Table 3).Table 3Share of public finance managed by different segments of the health systemBudget managementYear2002/032003/042004/05%%%Ministry of Health60.249.551.6National AIDS Commission1.83.511.9CHAM4.22.94.2Other NGOs4.37.96.4Donors10.62010.9Other18.916.215(Source: Malawi 2004/05 National Health Accounts – draft copy (September 2006). Lilongwe: Malawi Ministry of Health)Health services are provided by a multiplicity of providers. Of 'formal' health facilities, 60% are government-run; and 26% are mission facilities (mainly found in the rural areas). There is a small private-for-profit health sector (including three private hospitals) limited mainly to urban areas, as well as services provided by private companies for their employees. There is also a substantial traditional health sector. Nearly a quarter of deliveries are attended by a traditional birth attendant.Mission facilities tend to operate independently of each other but within a loose association called the Christian Health Association of Malawi (CHAM). A formal agreement exists with the Ministry of Health whereby most of the CHAM workforce is paid from the government payroll. Other providers include islamic health facilities, NGOs, grocery stores, pharmacies and community-based distribution agents for contraception. The share of total health care expenditure in 1998/9 amongst different providers is shown in Figure 4. Since then, NGO health care provision has expanded, particularly NGOs providing HIV/AIDS services. There are also a number of clinical research projects, particularly related to HIV/AIDS in the health care system – these provide services to research subjects but also consume a significant number of the country's scarce skilled health workforce (see Figure 4).Figure 4Health expenditure in Malawi by provider sector, 1998/9 FY. (Source: Government of Malawi, Ministry of Health and Population: Malawi National Health Accounts: a broader perspective of the Malawian Health Sector, 2001).In theory, health care providers in Malawi are organized according to a system of five 'zones' and 28 'health districts'. Each district is supposed to have an integrated health plan that incorporates the public sector, CHAM facilities and NGO providers. In practice, this does not always happen. Zonal offices which are supposed to provide support and supervision to district level services are relatively new and do not yet have the capacity to effectively support health districts. And in many districts, public, CHAM, NGO and private providers operate independently of each other.Presently, local government assemblies provide a small amount of health services. However, there are plans to devolve primary health care provision to local assemblies, including the transfer of budgets and human resource employment responsibilities.Human resourcesAs noted in the introduction, the total density of doctors and nurses (including midwives) in Malawi is 0.61 per thousand population, a shade higher than the threshold of 0.5 that defines the 10 worst served sub-Saharan African countries. As in other countries, the national average masks extreme inequities of provision within the country. In November 2004, 15 out of Malawi's 26 districts had less than 1.5 nurses per facility, and five had less than one. Only 13% of all health facilities had 24-hour midwifery coverage. Of 28 600 health worker posts in Ministry of Health (MoH) and CHAM facilities in 2005, about 38% were vacant [22]. Half of Malawi's doctors work at one of four central hospitals (although this partly due to deployment of newly qualified doctors to the central hospitals for the period of internship).The HRH situation was described in April 2004 by the MoH as \"dangerously close to collapse\" and as a \"major, persistent and deepening crisis\" [23]. An independent review of a safe motherhood project concluded that in spite of \"extensive staff training and support\" to midwives, problems with staff retention would remain an important obstacle to increasing coverage of births by skilled attendants [24].Three notable features of the health workforce in Malawi are the extensive use of clinical officers, medical assistants and about 4500 community-based health surveillance assistants (HSAs). Clinical officers receive four years of training and provide a range of medical services, including diagnosis and treatment, surgery and anaesthesia, and mending fractures. They form the cornerstone of hospital care in many rural areas. Medical assistants receive two years of training and mainly provide medical care in health centres and the outpatient departments of district hospitals. HSAs receive 10 weeks of training and are responsible for a variety of different tasks ranging from health promotion activities to TB defaulter tracing.There are several reasons for Malawi's health worker crisis. One is its low resource base which has made it difficult for the government to adequately fund the training, employment and retention of health staff. Even after establishing a medical school in 1991, Malawi produced only 20 doctors per year until 2005. Although it produced about 40–60 registered nurses and 300–350 enrolled nurses annually in the early 2000s [25], this is small compared to an establishment of 8,963 public sector nurses (including CHAM) [23].Another reason is HIV/AIDS. A 2002 study showed annual death rates of 2% among hospital health care workers [26]. Fear of exposure to HIV, particularly as shortages of gloves and other supplies hampers adherence to universal precautions, is also said to have contributed to staff leaving the sector [27]. Staff time is also lost to funeral attendance, care of sick family members and prolonged periods of illness. The increased workload caused by HIV/AIDS has also contributed to further demotivation, although according to local informants, the ability to treat patients with antiretroviral therapy is said to have improved staff morale.Staffing problems are more acute in the public sector. Whereas 20 years ago, public sector health worker salaries were considered attractive, wages for civil servants in Malawi have not kept up with rising consumer prices [28]. Job opportunities in the better-paid private sector (including NGOs and research institutions) and abroad, particularly in the UK, have been another potent 'pull factor'.Other reasons staff leave the public sector include poor working conditions; infrequent supervision and support; the lack of essential drugs, supplies and equipment; limited career progression opportunities; unequal access to training; an unclear deployment policy; and poor housing [29]. A study conducted in early 2006 identified up to 740 'inactive' professional health workers, including 469 nurses and 164 Clinical Officers, who had either resigned or retired from the health sector [30].The Emergency Human Resources ProgrammeMalawi has implemented a variety of initiatives to solve its health worker shortages over the years. However, it was only after Peter Piot, Executive Director of UNAIDS, and Suma Chakrabarti, Permanent Secretary of the UK Department for International Development (DfID), visited Malawi in 2004 and witnessed first hand the hopeless staffing situation of many facilities that a substantial human resources plan was pulled together. The result was a shift from piecemeal donor support to a comprehensive six-year \"Emergency Human Resources Programme\" (EHRP).Costed at US$272 million, with major funding from DFID and some from the Global Fund, the EHRP aims to raise Malawi's staffing levels (see Table 4) to a point where it could deliver the EHP (the planned targets do not therefore cater for the additional staff needed to provide antiretroviral therapy services). Although the EHRP would significantly boost staffing levels, the targets still fall short of the WHO-recommended minimum (on a rough estimate the EHRP would increase the total doctor and nurse density to 1.51 compared to the 2.28 threshold used by the 2006 WHR to define a 'critical shortage') (Table 4).Table 4Selected EHRP staffing targets (F/Y 2005–2006 Stock Indicator)CategoryCombined Ministry of Health & CHAMEHRP TargetFilledVacancy Rate (%)Physician/Specialist43316263%Nurse (all categories)8,4403,41660%Clinical Officer1,4051,03326%Medical Assistant1,50049167%Radiography/Technician2705879%Pharmacy/Technician26913450%Medical Laboratory Technician50718264%Environmental Health Officer1,66222387%Dental Technician/Therapist47013871%Physiotherapy1682287%Medical Engineering602460%Health Surveillance Assistant11,0004,66458%(Source: Government of Malawi, Ministry of Health (July 2006). Strategic human resources for health framework for the health sector)The EHRP takes a five-pronged approach:• Improving incentives for recruitment and retention of public sector and CHAM staff through a 52% salary top-up for 11 professional and technical cadres, coupled with a major initiative to recruit and re-engage qualified Malawian staff.• Expanding domestic training capacity, including doubling the number of nurses and tripling the number of doctors in training.• Using international volunteer doctors and nurse tutors as a short-term measure to fill critical posts while Malawians are being trained.• Providing technical assistance to bolster Ministry of Health (MoH) capacity in human resources planning, management and development.• Establishing robust human resources monitoring and evaluation capacity.In addition, the programme explicitly recognises the importance of improving policies on postings and promotions; training and career development; and incentives for deploying staff to underserved areas (which includes a major effort to improve staff housing). Technical assistance to the MoH in the form of human resources experts was therefore arranged. The government has also introduced a period of compulsory public health service for enrolled nurses trained at public expense.Contrary to initial fears, other public servants did not protest at the improved pay for health workers partly because of the careful way in which the government and others had made the case for higher pay for health workers [31]. However, any further improvements to the pay and working conditions of health workers are likely to be resisted without improvements for other civil servants.Since its implementation, anecdotal reports indicate that the salary rise had helped stem the flow of staff, particularly nurses, out of the public sector [31]. In addition, by the last quarter of 2005, 591 'inactive' staff had been recruited and more than 1,100 staff had been promoted (mostly nurses whose promotions had been blocked by civil service rules following a change to the nursing curriculum).The number of health professionals trained annually increased from 400/year in 2004 to over 1000/year in 2006. The College of Medicine increased its first-year Medical Doctor intake for 2005 to 60 students [22]. By mid-2006, health-training institutions were running at full capacity, albeit with a need to improve tutor: student ratios. To further increase the output of nurse training institutions, proposals exist to reduce the length of time required for basic nurse training from four to three years (longer than in most other African countries). In 2006, 51 expatriate doctors and 15 nurse tutors were scheduled to be in post [31].However, a recent evaluation of the EHRP in 2006 concluded that there were still difficulties in attracting tutors, doctors and nurses and that the EHP would not succeed if \"radical action is not taken to dramatically improve retention rates\", particularly in rural areas [32]. Another illustration of on-going problems was the observation that although expatriate doctors had been recruited successfully through the UN Volunteer Programme, in 2004/05 and 2005/06 less than eight medical graduates had joined the MoH whilst several other junior doctors had resigned [33].According to Medecins Sans Frontieres (MSF), in Chiradzulu district, there were 50 nurses working at the district hospital in 2006; that number had dropped to 28 by 2007 [34]. MSF also noted the experience of retired nurses who had been attracted to return to the workforce having trouble getting contracts and payment due to administrative delays.One problem was that the promised 52% salary top-up was not translated into a 52% increase in take-home pay because of changes to the tax and allowance structure of public sector health workers. Furthermore, in spite of the salary top-ups, non-government employers still offer much better rates of pay, particularly for scarce health worker cadres such as doctors, laboratory technicians and pharmacists.The fragmented and competitive provider market, coupled with the pressure on funders and policy makers to achieve ambitious coverage targets, has caused the labour market to become extremely uneven. Scarce skills appear to be concentrated in urban areas and in NGO/research projects that are able to offer higher remuneration. According to MSF, external financing is also associated with workshops and training programmes which public health workers are paid with per diems and stipends to attend. A five-day training workshop can increase a nurse's basic monthly salary by 25–40% [34]. Although training workshops are necessary, the competition for stipends can disrupt service delivery and increase absence from facilities.HIV/AIDS and the provision of antiretroviral therapyIn spite of its significant health systems constraints, Malawi has made exceptional progress in expanding access to ART. At the end of 2006, there were about 60 000 people on treatment in the country, with plans to expand coverage to 245 000 people by 2010.This progress is argued to have been achieved because of several factors [35]: A strong rights-based international advocacy movement Earmarked funding for antiretroviral therapy services from a range of donors. Support from international NGOs and research organisations to deliver ART services Strong technical leadership and management within the Ministry of Health A vertical management and delivery system which has included:◦ dedicated ART training programmes for various cadres of health workers (see below)◦ A stand-alone system for financing, procuring and distributing antiretroviral therapy drugs. This involves drugs procured by UNICEF from India being flown to Copenhagen where they are individually packed for each ART clinic, and then flown to Malawi where they are couriered to each ART clinic.◦ A stand-alone information system to enable high-quality monitoring and evaluation◦ Quarterly supervision and support visits to all ART clinics A 'low-resource approach' which includes using a single first-line and second-line regimen for all patients and providers; using clinical staging to determine eligibility for treatment (not CD4 counts); using fixed-dose combination tablets; and using clinical signs only to monitor treatment response.Under the direction of the HIV/AIDS unit within the MoH, an agreement has been reached that ART providers will be supplied with government-procured drugs, whether in the public or private sector, provided they attend a 5-day training course and formal assessment. In the private sector, in addition to paying private consultation fees, patients pay a fee of MK 500 (at time of writing, US$ 1 = MK 140) per month for the medicines, of which MK 200 is retained by the private provider and MK 300 is paid into a revolving fund managed by the Malawi Business Coalition Against HIV/AIDS which is then remitted to the National AIDS Council. The cost of ART on the government procurement scheme is approximately MK 1820 per month, although this excludes the costs of supply and distribution logistics [36].Before new ART sites are established, those responsible for establishing the sites and providing care must also spend two weeks attached to one of the specialist HIV centres within Malawi after completing the 5-day training course. Through the provision of subsidised medicines and using this model of structured training, the government has been able to harness the private sector to support the national ART programme.Human resource plans to further expand ART coverage involve four main strategies [35]:1) minimizing the health worker: patient ratio by changing the requirement for all patients to be seen by a clinician when they come for repeat prescriptions.2) 'task-shifting' to enable nurses to diagnose and prescribe ART and 'lower' cadres of health workers (in particular HSAs) to dispense ART. Plans exist to overcome legal and professional restrictions on prescribing and dispensing, and to train and equip HSAs with the competencies to provide ART drugs; keep accurate patient records; and question carers and patients so that an appropriate treatment regime and referral pattern can be established. [However, it should be noted that this is being resisted by the Pharmacy, Medicines and Poisons Board]3) increasing the number of health workers involved in the ART programme by including volunteers/unpaid workers.4) decentralizing management and supervision to zonal and district health management structures.In Thyolo district, MSF has been working with the government to provide more than 10 000 people with ART by the end of 2007. One of its strategies has been to support 600 volunteer community home-based caregivers to assist community nurses with the management of common HIV-related conditions, support people on ART, and trace defaulters [37]. Nurses are also being used to manage 'stable patients' (defined as non-pregnant adults who have been on first-line treatment for at least one year with no complications or adherence problems).However, as Malawi contemplates the further expansion of antiretroviral therapy (whilst sustaining its current gains) within the context of limited resources and many shortfalls in the provision of other essential health services, there are concerns that these other services could be harmed. In addition, the intention to decentralize new responsibilities to zonal and district offices might compromise this management capacity which is already struggling to oversee and support other health programmes.The external evaluation of Malawi's ART programme in 2006 noted concerns that antiretroviral services resembled 'islands of excellence in a sea of problems' [35]. While the ART programme's achievements were impressive, other services (including the prevention of vertical transmission) showed signs of stagnation. One contrast was the excellent supply of ART drugs compared with the abysmal supply of other essential health commodities; another was the plans to up-scale paediatric ART when it was clear that the country's programme to reduce vertical transmission had stalled. It was also noted that the ART programme's focus on individual treatment had under-emphasised the potential for treatment services to act as an engine for HIV prevention.However, the ART programme could also impact positively on the health system by, for example, helping keep HIV-positive health workers healthy and preventing facilities from being overwhelmed by the needs of people dying from AIDS. In addition, the political and civic energy and additional resources directed at the scale up of ART provides an opportunity to strengthen health systems. For example, the impetus to reduce vertical HIV transmission can be harnessed to improve the quality of ante-natal and obstetric care as a whole.The recent introduction of eight paediatricians from the United States to help increase coverage of paediatric ART is another example of how the current international focus on AIDS treatment could be harnessed to strengthen the health system as a whole. As well as increasing paediatric ART coverage, these physicians could be deployed to support the improvement and expansion of other child health services.ConclusionMalawi is one of a few countries in extreme crisis in relation to both human resources and HIV. Despite this, it is making remarkable progress both in tackling the causes of human resource problems and in providing antiretroviral therapy. The case highlights how both synergies and conflicts between the two strategies have been realised in this context out of the many possible configurations that could be theoretically predicted.Among the evident synergies is the contribution of the EHRP to increasing the availability of staff for the expanding ART programme both in total and in underserved areas. This is consistent with the growing acknowledgement that the basic human infrastructure of health systems in SSA must be strengthened if global ART coverage targets are to be met.However, while Malawi has been fortunate enough to receive donor support for its EHRP, it is clear that more needs to be done. Donors and the government still need to secure additional resources to increase staff recruitment and training, and to improve retention if WHO's minimum HRH standards are to be met. In addition greater attention will need to be paid to the dynamics of the domestic labour market and the disparities between private and public sector remuneration if Malawi is to ensure a more rational and needs-based deployment of health workers across the country.The impact of external project funding on workforce balance has been suggested to lead to the need for health workforce impact assessments as part of project appraisal: 'It could be envisaged that at country level, public and private health services, NGOs and international agencies that would like to start up a new programme or activity would have to demonstrate the impact of their plan on the current health workforce to the Ministry of Health. Similarly, organizations applying for funding at international donor agencies would be asked the same' [38].The adoption of a low-resource model of ART provision, underscored by task shifting and the use of clinical officers and nurses to diagnose and treat AIDS patients is also consistent with protecting staff availability to also meet other demands on the health system. Moreover, the effective delivery of ART should reduce demands on the health care system for the treatment of opportunistic infections and end-of-life care, although this may only be a short term impact. As the ART programme matures, the number of treatment failures to first-line regimens will grow and unless there is the capacity to fund and supervise the use of second-line treatment, the health care system could see a 'rebound effect' of returning AIDS patients.Another notable achievement of the Malawi ART programme has been its ability to incorporate private sector providers into the national ART programme. This has been achieved through a quid pro quo arrangement whereby private sector providers agree to adopt national treatment and monitoring policies in exchange for medicines that are mainly paid for by the public purse.The potential for synergy in the strengthening of drug supply systems has not yet been realised. The procurement and supply system for ARVs described earlier operates in isolation from the procurement and supply system for other medicines and commodities, which includes a number of other stand-alone, vertical systems.Among the evident conflicts are the demands placed on service delivery by additional in-service training activities and the conflicts between dedicating resources to ensuring the excellence of the antiretroviral therapy programme, and ensuring excellence in prevention – especially of mother-to-child transmission which could reduce the demands for a paediatric antiretroviral therapy programme. There is perhaps a more important tension between the patient-centred focus required for ART, and the community centred approach required for prevention programmes that target the broader determinants of HIV infection and other health problems. This may prove more difficult to resolve.Planning for the rapid scale-up of antiretroviral therapy whilst simultaneously strengthening health systems and delivering the broader EHP will require prioritizing amongst the different health care needs and careful consideration of coverage targets and timeframes set by the antiretroviral therapy programme because it takes time (not just resources) to develop positive synergies between antiretroviral therapy scale-up and health systems strengthening. It will also require the right balance between the use of 'dedicated' antiretroviral therapy cadres of health workers and an antiretroviral therapy programme operated through 'generalist' health workers capable of providing comprehensive care. The former may result in faster and more effective antiretroviral therapy coverage, but the latter may be more sustainable and be less harmful to other health care services.A greater emphasis on sustainability and health systems strengthening may compromise the speed of up-scaling in the short-term but could derive greater benefits in the long-term by, for example, ensuring a high level of treatment adherence, keeping patients on first line treatment for longer and deferring the use of more expensive second-line treatments.AbbreviationsART: antiretroviral therapy; CHAM: Christian Hospital Association of Malawi; EHP: essential health package; EHRP:  Emergency Human Resources Programme; GDP: gross domestic product; HSA: health surveillance assistants; MSF: Medecins Sans Frontieres; NAC: National AIDS Commission; NGO: non-government organisations; VCT: voluntary counselling and testing; WHO: World Health Organisation; WHR: World Health Report; SWAp: Sector Wide Approach.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsDM and BM drafted the paper on the basis of two separate papers they had individually authored. VM made significant contributions to the original draft with particular reference to the Malawi case study.\n\nREFERENCES:\nNo References"
+}

batch_10/PMC2533395.json

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+{
+    "id": "PMC2533395",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533395\nAUTHORS: Eric Jeziorski, Brigitte Senechal, Thierry Jo Molina, Francis Devez, Marianne Leruez-Ville, Patrice Morand, Christophe Glorion, Ludovic Mansuy, Joel Gaudelus, Marianne Debre, Francis Jaubert, Jean-Marie Seigneurin, Caroline Thomas, Irene Joab, Jean Donadieu, Frederic Geissmann\n\nABSTRACT:\nBackgroundLangerhans cell histiocytosis (LCH) is a rare disease that affects mainly young children, and which features granulomas containing Langerhans-type dendritic cells. The role of several human herpesviruses (HHV) in the pathogenesis of LCH was suggested by numerous reports but remains debated. Epstein-barr virus (EBV, HHV-4), & Cytomegalovirus (CMV, HHV-5) can infect Langerhans cells, and EBV, CMV and HHV-6 have been proposed to be associated with LCH based on the detection of these viruses in clinical samples.MethodologyWe have investigated the prevalence of EBV, CMV and HHV-6 infection, the characters of antibody response and the plasma viral load in a cohort of 83 patients and 236 age-matched controls, and the presence and cellular localization of the viruses in LCH tissue samples from 19 patients.Principal FindingsThe results show that prevalence, serological titers, and viral load for EBV, CMV and HHV-6 did not differ between patients and controls. EBV was found by PCR in tumoral sample from 3/19 patients, however, EBV small RNAs EBERs –when positive-, were detected by in situ double staining in bystander B CD20+ CD79a+ lymphocytes and not in CD1a+ LC. HHV-6 genome was detected in the biopsies of 5/19 patients with low copy number and viral Ag could not be detected in biopsies. CMV was not detected by PCR in this series.Conclusions/SignificanceTherefore, our findings do not support the hypothesis of a role of EBV, CMV, or HHV-6 in the pathogenesis of LCH, and indicate that the frequent detection of Epstein-barr virus (EBV) in Langerhans cell histiocytosis is accounted for by the infection of bystander B lymphocytes in LCH granuloma. The latter observation can be attributed to the immunosuppressive micro environment found in LCH granuloma.\n\nBODY:\nIntroductionLangerhans cell histiocytosis (LCH, a.k.a. histiocytosis X), is a rare disease that affects mainly young children, and features granulomas consisting of Langerhans-like cells (LC), mixed with macrophages, eosinophiles, multinucleated giant cells, and lymphocytes, that can be found within various tissues [1], [2].The presence of LC in granuloma is a key diagnostic feature of LCH. LC are members of the dendritic cell (DC) family, that trigger and shape immune responses, and the pathophysiology of LCH is likely to involve immune mechanisms (reviewed in [3], [4]). We have previously reported that LC found in LCH granuloma were phenotypically and functionally immature/semi-mature LC [5]. Immature/semi-mature DCs are believed to be prone to induce regulatory T cells, that inhibit polyclonal T cell responses and promote tolerance [6], [7], [8], [9]. The accumulation of immature LC in LCH granulomas was associated with the expansion of FoxP3+ CD25 + CD4+ regulatory T cells both in granuloma and in the blood of patients [10]. Therefore local and general immunosuppression, which favors reactivation of herpes-virus infection, may be a feature of LCH.Environmental agents and viruses, in particular Epstein-Barr virus (EBV), or vaccination, have been proposed to trigger, or to play a role in the pathogenesis of the disease [11], [12]. Herpesviruses are DNA viruses responsible for persistent infection. EBV is the etiological agent of several malignancies [13], [14], and EBV & Cytomegalovirus (CMV) are responsible for hemophagocytic syndromes in human with several inherited immunodeficiencies [15], [16]. EBV has been reported to infect monocytes and Langerhans cells (LC), during the natural course of infection in human [17], [18]. Infection with EBV has been reported to be associated with LCH, to represent a possible etiology, and/or to contribute to its pathophysiology in some studies [11], [12], [19]. However, other studies failed to replicate these findings, and the possible causative role of EBV in LCH is debated [20], [21], [22]. CMV can also infect DC and LC [23], [24], [25], and one single study reported CMV detection in lesional LC in one third of 29 patients by immunohistochemistry, in situ hybridization, and PCR [26]. HHV-6 infects mainly T cells, but is also reported to infect myeloid cells [27], [28] and HHV-6 DNA or immunoreactivity was detected in lesions of 50% to 75% of patient with LCH [29], [30], however, control studies performed by the same group concluded that the prevalence of HHV-6 in the tissue of LCH patients is the same as that found in tissue from individuals without disease [31].Sero-epidemiological studies have been useful to demonstrate the role of EBV in Burkitt lymphoma and Hodgkin disease, when high antibody titers to EBV structural antigens (VCA) have been associated to the risk of developing Burkitt's lymphoma and Hodgkin diseases [32], [33]. However, to our knowledge no sero-epidemiological study have been conducted in LCH. The present study was therefore designed to investigate the role of EBV, CMV, and HHV-6 using two methods. First, we performed a case-controlled sero-epidemiological study to investigate a relationship between the onset of LCH in young children and the antibody response to infection with EBV, CMV, or HHV-6, and second we searched for the presence of viruses in the serum of patients, and in Langerhans cells in tumor samples, by PCR and, when positive, we investigated the cellular target of the viruses by immunolabeling and in situ hybridization. Results ruled out an epidemiological association between these herpes-virus and LCH.ResultsPrevalence of EBV, CMV, and HHV-6 infection as a function of age and of clinical presentation of disease. The presence of antibodies against EBV, CMV and HHV-6 was investigated by ELISA in the serum of 78 children diagnosed with LCH and 206 age matched controls. To avoid contamination by maternal IgG, only children six month-old and older where studied for the presence of specific antibodies in serum. Twenty six children with LCH (33.3%) and 94 controls (47.1%) tested seropositive for EBV. No significant difference was observed between patient and control groups when matched for age (Table 1). Similarly, no significant difference was observed with the control group when patients were grouped by disease stage/clinical presentation (Table 2). Similar results were observed for CMV and HHV-6, and the prevalence of both infections in children with LCH were comparable to their prevalence among age-matched controls (Tables 2,and 3).10.1371/journal.pone.0003262.t001Table 1Prevalence of EBV, CMV, and HHV-6 infection in LCH patientsControlsLCH totalLCH1LCH2LCH3EBVNon-infectedn10952281212%52.966.765.163.275Infectedn97261574%41.133.334.936.825Totaln20678431916%100100100100100Test Fisher's exact0.475CMVNon-infectedn13158321313%64.275.376.268.481.2Infectedn7218963%35.323.421.431.618.8\nUnknown*\nn11100%0.51.32.400Totaln20477421916%100100100100100Test Fisher's exact0.282HHV-6Non-infectedn3315555%1.419.511.926.331.2Infectedn16162371411%80.180.588.17.768.7\nUnknown*\nn70000%3.50000Totaln20177421916%100100100100100Test Fisher's exact0.424Unknown*: IgG value are in the ‘grey zone’ (see methods)LCH1: single system, no risk organsLCH2: multisystem, no risk organsLCH3: risk organs10.1371/journal.pone.0003262.t002Table 2Prevalence of EBV, CMV, and HHV-6 infection as a function of ageAge 0.5 to 5Age 5 to 10Age 10 to 20ControlsLCHControlsLCHControlsLCHEBVNon-infectedn79351911116%71.885.435.845.825.646.1Infectedn3163413327%28.214.664.154.274.453.8Totaln1104153244313%100100100100100100Test Fisher's exact0.0930.5770.378CMVNon-infectedn743429132711%69.48554.754.262.884.6Infectedn3362310162%30.61543.441.737.215.4Unknown*n001100%001.94.200Totaln1074053244313%100100100100100100Test Fisher's exact0.0620.820.186HHV-6Non-infectedn181450101%17359.4023.87.7Infectedn842647243012%79.26588.710071.492.3Unknown*n401020%3.801.904.80Totaln1064053244213%100100100100100100Test Fisher's exact0.0370.2170.35610.1371/journal.pone.0003262.t003Table 3Prevalence of HHV-6 infection in children before the age of 5 yearsAge 0.5 to 1Age 1 to 1.5Age 1.5 to 2Age 2 to 5controlsLCHcontrolsLCHcontrolsLCHcontrolsLCHNon-infectedn58024094%35.766.7033.330.8014.821Infectedn94174934915%64.333.395.466.769.210080.379Unknown*n00100030%005.60004.90Totaln14121861336119%100100100100100100100100Test Fisher's exact0.2380.0540.5290.663Serum IgG titers directed against EBV, as well as CMV and HHV-6, did not differ between patients and controls. Titers of EBV antibodies have been shown to differ from controls in several diseases linked to EBV [14], [34]. IgG VCA titers, and in some cases anti-Epstein-Barr nuclear antigen (EBNA)1 antibody, are consistently higher in patients with nasopharynx carcinoma or Hodgkin disease than in control populations [14]. Lower EBNA1-IgG antibody titer is also considered as a possible serological sign for a defective control of the persistent latent EBV carrier state. In the present study, both VCA IgG and EBNA IgG titers were found to be similar in patients with LCH in comparison with controls, when matched for age, or when grouped by disease stage/clinical presentation (Figure 1A–E). Anti HHV-6 or anti CMV IgG titers were also not different between patients and the control group (data not shown)10.1371/journal.pone.0003262.g001Figure 1VCA-IgG and EBNA-IgG titers (UA/ml) in patients and controls with past EBV infection.VCA-IgG (A, B, C) and EBNA-IgG titers (D, E, F) (UA/ml) were determined as indicated in methods, and patients were compared to controls without stratification (A, D), and after stratification based on age (B, E), or disease extension (C, F).Prevalence of detectable serum viral load for EBV, CMV, and HHV-6. Defective control of infection by Herpesviruses such as EBV, CMV and HHV-6 ultimately results in replicative infection and viremia [35]. Serum viral load during CMV infection is a sensitive technique, but in the present study, out of 83 patients and 236 age-matched controls, 0% of patient and 3% of controls (n = 7) had detectable CMV serum viral load (table 4) indicating the very low indidence of active infection. Serum viral load during EBV infection is considered to be less sensitive than during CMV infection [36], nevertheless we found that only 1.2% of patients (n = 1) and a similar percentage (0.9% of controls, n = 2) had a detectable EBV viral load in serum (Table 4). These results are consistent with anti-VCA IgG titers, which were found similar in patients and controls (see figure 1), since EBV viral load was shown to be correlated with anti-VCA IgG titer [34]. The significance of the detection of HHV-6 in the serum is still discussed [37], [38], but again only 3.6% of patients (n = 3) and 2.5% of controls (n = 6) had a detectable HHV-6 serum viral load, and there was no difference between groups as per fischer exact's test (Table 4).10.1371/journal.pone.0003262.t004Table 4Prevalence of detectable serum viral load for EBV, CMV, and HHV-6PatientsControlsTotalEBVNegative82234316%98.899.199.1Positive123%1.20.90.9Total83236319%100100100Fisher's exact = 1.0001-sided Fisher's exact = 0.596CMVNegative83229312%10097.97.8Positive077%032.2Total83236319%100100100Fisher's exact = 0.1971-sided Fisher's exact = 0.118HHV-6Negative80230310%96.497.597.2Positive369%3.62.52.8Total83236319%100100100Fisher's exact = 0.7011-sided Fisher's exact = 0.428Detection of EBV, CMV, and HHV-6 DNA in biopsy samples from LCH granuloma. We then investigated whether Herpesviruses were present in LCH granuloma, in the absence of overt viremia. As shown in table 5, EBV DNA was found in 15% of biopsy samples examined (3/19 patients, #1, 9, & 15), and HHV-6 DNA in 26% of biopsy samples examined (5/19 patients). CMV DNA was not detected in this series. EBV and HHV-6 viral loads were low with a median of 400 copies/106 cells except for patient #5. Patient #5 display a very high HHV-6 load in the biopsy and in the blood over time (data not shown) compatible with the detection of a chromosomally integrated HHV-6 DNA [39].10.1371/journal.pone.0003262.t005Table 5Detection of EBV, CMV, and HHV-6 DNA in biopsy samples from LCH granuloma.PatientstagesexHHV-6EBVCMV1LCH1F-6 103\n-2LCH1M8 102\n--3LCH1M---4LCH1F6 101\n--5LCH1M2 106\n--6LCH1F---7LCH1M---8LCH1F---9LCH1F-1.7 105\n-10LCH1F---11LCH1M---12LCH1M---13LCH1M---14LCH1F---15LCH1M-4 102\n-16LCH2M4 102\n--17LCH3M---18LCH3M---19LCH3M4 102\n--Positive results are expressed as the number of viral genome copies per million cells.Therefore, our data support previous results showing the presence of EBV or HHV-6 in a subset of LCH granuloma. However, since HHV-6 and EBV are responsible for persistent infection of T cells and B cells respectively, the detection of virus DNA in LCH samples could be attributed to infection of bystander cells [21].We therefore investigated whether EBV infects bystanders lymphocytes, macrophages, and Langerhans cells in samples from patients #1, 9, & 15, which tested positive by PCR, using in situ hybridisation with a probe against EBV EBERs RNA on paraffin sections of LCH granuloma, followed by immunohistochemistry with antibodies against B-cells (CD20 & CD79a), T-cells (CD3), macrophages (CD68), or Langerhans cell (CD1a) antigens on the same tissue sections. EBV positive cells were always found in areas rich in B cells, and were labeled with either CD20, or CD79a antibodies (Figure 2), while no EBER+ cell labelled with CD1a, CD3, or CD68 cell was observed. These data indicate that EBV infects bystander B cells, which are present in a subsets of LCH granuloma [12]. Although positive controls were obtained, we did not observed immunoreactivity against HHV-6 in samples positive for HHV-6 DNA by PCR (data not shown), therefore the cellular target of HHV6 in LCH granuloma was not identified.10.1371/journal.pone.0003262.g002Figure 2Detection of EBV-infected B cells by in situ hybridization combined with immunohistochemistry.Granuloma serial sections were stained for CD1a (upper left) or for CD20 (middle left) by immunohistochemistry or for EBERs by in situ hybridisation (upper right, arrows indicate EBER positive cells). Combined detection of CD20 or CD79a with EBERs shows that EBV-infected cells are B cells (arrows).DiscussionThe present study is the largest case-controlled sero-epidemiological study performed in this disease. The results argue against an epidemiological association between LCH and EBV, CMV, or HHV-6 infection.Further investigation of the cellular localization of the viruses in LCH tissue samples from 19 patients indicated that, when present, EBV infected bystander lymphocytes and not Langerhans cells. We think that our results therefore resolve the much debated issue of the role of EBV in the pathogenesis of LCH [19], [21], [22], [30], [31]. Because EBV infected B cells are present in the blood of healthy seropositive individuals [40] at a low frequency in the order of 10-6\n[41] it is not unexpected to find a small number of EBV infected B cells in LCH granuloma, which contains B cells. In addition, because the growth of EBV infected B cells is under the control of CD8 cytotoxic T cells in healthy individuals [42], the regulatory T cell-rich environment of the LCH granuloma [10], may represent a sanctuary for such EBV infected B cells.Anti CMV IgG titers in patients and controls, and absence of detection of the virus by PCR in the serum and in biopsy samples strongly suggest that CMV is not associated with LCH.The case of HHV6 is more complicated. HHV-6 was detected by PCR in the serum of 3.8% of patients and 2.5% of controls with viral loads above 104 copies/ml in 5 out of 9 detected (data not shown). The prevalence of HHV-6 in patients and controls were similar to the reported prevalence of chromosomally integrated HHV6 among blood donors [39]. Therefore HHV6 detection may be related to the presence of chromosomally integrated HHV-6. HHV6 DNA was also detected by PCR in biopsies from 5/19 patients (26%), suggesting that HHV6, like EBV can be found in LCH granuloma. However the viral load was very low, and the virus was not detectable by immunochemistry suggesting that HHV-6 infection is quiescent. Together with our serological data and serum PCR, and in the light of the study of Glotzbecker et al, [31] who found a similar frequency PCR positive samples in LCH granuloma and control tissues, we hypothetize that HHV6 do not productivey infect LC in vivo and may be carried by bystander lymphocytes which are present in the granuloma.Our study did not identify a subset of patients with ‘herpes-viruses associated’ LCH. We did not found an association between EBV, CMV, or HHV-6 infection and patients when they were stratified by age, or by clinical stage.Among other herpes viruses, HHV-1 (HSV-1), HHV-2 (HSV-2), HHV-3 (VZV) and HHV-7 infections were never reported to be associated with LCH, and were not investigated in this study. Among herpesviruses, HHV-8 has been initially reported to infect dendritic cells, but several studies have excluded its association with LCH [43], [44].MethodsPatients & controlsThe diagnostic and inclusion criteria as well as the definition of the organs involvement for the French nationwide LCH survey have been described elsewhere [2]. Briefly, the extension of the diseases has been classified in three groups according to histiocyte society criteria: group 1: single organ extension, without risk organs group 2 mulsystem organ without risk organs and group 3 patients with risk organs i.e. lung and/or liver and/or spleen and/or hematological dysfunction. According to French bioethics laws, informed consent was signed if the patients participated and the database was approved by the French computer watchdog commission (CNIL certificate n° 99.087). Clinical information, radiological findings and extension were recorded, together with treatments received. Data monitoring, based on medical charts, was done by a clinical research associate who visited each center. Involvement of at least one new organ, as described elsewhere 2, was considered to define an LCH episode. Serum specimens were obtained, after written witnessed informed consent was obtained from the parents of all patients, from 83 pediatric patients with LCH included in the French LCH registry, following a research protocol approved by the ethics committee of the Nantes University Hospital (France, EU). Biopsies samples were also obtained from 19 patients with LCH included in the French LCH registry after written witnessed informed consent was obtained. Control serum samples were obtained from the children admitted to the outpatient unit of the Grenoble University Hospital (France, EU), according to institutional guidelines, and were matched for age with patient samples for statistical analysis.SerologyQualitative and quantitative analysis of specific Ig against EBV, CMV and HHV-6 were performed on serum samples from 83 patients and 235 controls using microplate ELISA kits: IgG anti-VCA (ETI-VCA-G, DiaSorin®), IgG anti-EBNA (ETI-EBNA-G, DiaSorin®), IgM anti-VCA (DiaSorin®,ETI-EBV-M), IgG anti-CMV(ETI-CYTOK-G PLUS, DiaSorin®) and IgG anti-HHV-6 (HHV-6 IgG EIA, Biotrin®). Patients and controls were classified in three categories according to the detection of specific IgG i/above the upper limit of the grey zone for “infected” status, ii/in the grey zone for “unknown” status, iii/below the lower limit of the grey zone for “uninfected” status.DNA extraction from frozen biopsies and serumDNA was extracted from sliced frozen biopsies using ALLPrep DNA/RNA minikit (Qiagen, Hilden, Germany). Total DNA was eluted with 200 ul of water and amplification of the beta-globin gene by real time PCR was used to evaluate total cell number per sample (Beta-globine PCR Kit, Roche Diagnostics®). DNA was extracted from 200ul of serum using Blood DNA mini kit and eluted in a volume of 100ul.Quantitative PCR for the detection of EBV, CMV and HHV-6ten ul of DNA extract were used to detect and quantify viral genomes by real time PCR assays. All positive PCR were run a second time for confirmation.EBV tyrosine kinase gene (TK) was amplified from biopsies DNA with T1 (5′-GGGGCAAAATACTGTGTTAG-3′)+T2 (5′-CGGGGGACACCATAGT-3′) primers and LC1 (5′-ATGTTTCCTCCCTCGCTTCTTCAG-fluo-3′)+LC2 (5′-ATGTTTCCTCCCTCGCTTCTTCAG-fluo-3′) probes. PCR were run on a Light Cycler. The EBV-negative DG75 cell line was used as a negative control. The EBV-positive Burkitt's lymphoma cell line “Namalwa” [45] harboring 2 copies of viral genome per cell is used as a standard for quantification. Detection limit is 2 copies of viral genome per amplification [46].CMV immediate early-1 gene (IE1) was amplified using forward 5′-GCAGACTCTCAGAGGAT-3′+reverse 5′-AGCGCCGCATTGAGGA-3′ primers and a 6-carboxyfluoresceine (FAM) -5′ ATCTGCATGAAGGTCTTTGCCCAGTACATT-3′ carboxytetramethyl rhodamine (TAMRA) probe. PCR were run on a ABI 7300 real time PCR system (Applied Biosystem).Quantification was obtained using a plasmid and the detection limit is 20 copies of viral genome per amplification [35].HHV-6 U65-U66 gene was amplified using forward 5′- GACAATCACATGCCTGGATAATG-3′+reverse 5′-TGTAAGCGTGTGGTAATGGACTAA -3′ primers and a 6-carboxyfluoresceine (FAM) -5′ AGCAGCTGGCGAAAAGTGCTGTGC-3′ carboxytetramethyl rhodamine (TAMRA) probe. PCR were run on a ABI 7300 real time PCR system (Applied Biosystem).Quantification was obtained using a plasmid and detection limit is 25 copies of viral genome per amplification [47].PCR results were expressed as the number of viral genome copies per million cells for biopsies and as the number of viral genome copies per ml of serum from peripheral blood.In situ hybridization & immunohistochemistryDetection of EBV EBERs RNA by in situ hybridization was performed first and was followed by immunohistochemistry with antibodies against B-cell, T-cell and Langerhans cell antigens. Five micrometers paraffin-embedded sections were mounted onto glass slides and pretreated in 0.4% pepsine HCl 0.2M, then hybridized with EBER PNA probes (DAKO), following the manufacturer's instructions, and revealed with NBT/BCIP after APAAP amplification. Immunohistochemistry was then performed using a streptavidin-biotin peroxidase method (LSAB2, DAKO) after microwave heating, revealed with AEC or DAB. 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@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533646",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533646\nAUTHORS: Emma J Eshuis, Willem A Bemelman, Ad A van Bodegraven, Mirjam AG Sprangers, Patrick MM Bossuyt, AW Marc van Milligen de Wit, Rogier MPH Crolla, Djuna L Cahen, Liekele E Oostenbrug, Meindert N Sosef, Annet MCJ Voorburg, Paul HP Davids, C Janneke van der Woude, Johan Lange, Rosalie C Mallant, Maarten J Boom, Rob J Lieverse, Edwin S van der Zaag, Martin HMG Houben, Juda Vecht, Robert EGJM Pierik, Theo JM van Ditzhuijsen, Hubert A Prins, Willem A Marsman, Henricus B Stockmann, Menno A Brink, Esther CJ Consten, Sjoerd DJ  van der Werf, Andreas WKS Marinelli, Jeroen M Jansen, Michael F Gerhards, Clemens JM Bolwerk, Laurents PS Stassen, BW Marcel Spanier, Ernst Jan Spillenaar Bilgen, Anne-Marie van Berkel, Huib A Cense, Henk A van Heukelem, Arnold van de Laar, Warner Bruins Slot, Quirijn A Eijsbouts, Nancy AM van Ooteghem, Bart van Wagensveld, Jan MH  van den Brande, Anna AW van Geloven, Karien F Bruin, John K Maring, Bas Oldenburg, Richard van Hillegersberg, Dirk J de Jong, Robert Bleichrodt, Donald L van der Peet, Pascal EP Dekkers, T Hauwy Goei, Pieter CF Stokkers\n\nABSTRACT:\nBackgroundWith the availability of infliximab, nowadays recurrent Crohn's disease, defined as disease refractory to immunomodulatory agents that has been treated with steroids, is generally treated with infliximab. Infliximab is an effective but expensive treatment and once started it is unclear when therapy can be discontinued. Surgical resection has been the golden standard in recurrent Crohn's disease. Laparoscopic ileocolic resection proved to be safe and is characterized by a quick symptom reduction.The objective of this study is to compare infliximab treatment with laparoscopic ileocolic resection in patients with recurrent Crohn's disease of the distal ileum with respect to quality of life and costs.Methods/designThe study is designed as a multicenter randomized clinical trial including patients with Crohn's disease located in the terminal ileum that require infliximab treatment following recent consensus statements on inflammatory bowel disease treatment: moderate to severe disease activity in patients that fail to respond to steroid therapy or immunomodulatory therapy. Patients will be randomized to receive either infliximab or undergo a laparoscopic ileocolic resection. Primary outcomes are quality of life and costs. Secondary outcomes are hospital stay, early and late morbidity, sick leave and surgical recurrence. In order to detect an effect size of 0.5 on the Inflammatory Bowel Disease Questionnaire at a 5% two sided significance level with a power of 80%, a sample size of 65 patients per treatment group can be calculated. An economic evaluation will be performed by assessing the marginal direct medical, non-medical and time costs and the costs per Quality Adjusted Life Year (QALY) will be calculated. For both treatment strategies a cost-utility ratio will be calculated. Patients will be included from December 2007.DiscussionThe LIR!C-trial is a randomized multicenter trial that will provide evidence whether infliximab treatment or surgery is the best treatment for recurrent distal ileitis in Crohn's disease.Trial registrationNederlands Trial Register NTR1150\n\nBODY:\nBackgroundCrohn's disease is an inflammatory bowel disease that affects the entire gut, but mostly the terminal ileum of the small bowel is involved. Due to the chronic inflammation the affected bowel segment is scarred and may become stenotic. Although medical treatment aims to reduce the inflammation, many patients eventually will have surgery because of obstructive complaints [1].Primary medical treatment is still considered the preferred treatment because of the potential morbidity associated with surgery. Furthermore, medical treatment might avert surgery. Medical therapy consists of remission induction by a short course of steroids most often followed by maintenance therapy with immunomodulating agents. Recurrence of disease activity is primarily treated with steroids. Frequent disease exacerbations and steroid dependency are an indication for treatment with infliximab. Infliximab is a chimeric anti-TNF monoclonal antibody against tumor necrosis factor, an important proinflammatory cytokine in Crohn's disease. Treatment with this biological is effective in inducing and maintaining response and remission in patients with moderate to severe Crohn's disease.Infliximab therapy once initiated is best continued at 8 weeks intervals, although interval therapy is often used to reduce costs and to avoid the risks of long-term immune suppression. Major drawbacks of medical therapy are long-term use of medication with associated impairment of quality of life, morbidity and high costs. Furthermore, infliximab treatment is an open-ended medical treatment: it is unclear for how long therapy should be continued. Interrupting the treatment is undesirable since it is associated with loss of response due to anti-infliximab antibody formation [2-4]. It remains unclear in how many patients with recurrent Crohn's disease surgery can eventually be avoided [1] Thus, patients with recurrent Crohn's disease encompass a heterogeneous group of patients some of which will respond to (long-term) medical treatment whereas in others surgery cannot be averted by medical treatment.It is well established that an ileocolic resection is an effective and low morbidity operation resulting in a quick relieve of complaints and fast restoration of quality of life. Most frequent complications requiring reoperation are anastomotic dehiscence and intra-abdominal abscess. In several publications analyzing safety of laparoscopic ileocolic resection, the percentage of complications requiring reoperation varied from 0 to 7.6% [5]. After ileocolic resection, medication can be stopped or limited to prophylactic medication when indicated [6]. The length of loss of small bowel is generally limited and averages 20–25 cm in patients who had surgery for obstructive symptoms refractory to medical treatment. Long-term surgical recurrence occurs in 20–25% over an 8–9 years period in patients refractory to medication [7,8].Patients are generally young and in the middle of building their socioeconomic life. Disease activity with its associated complaints and long-term therapy have a pronounced effect on quality of life characterized by sick leave and non-attendance of social activities [9,10]. Patients that have a clinical recurrence after medical treatment can be considered as patients having a more severe type of the disease. To date consensus statements offer either treatment with infliximab or surgical resection in limited disease, because no comparative studies on the two alternatives exist. It can be hypothesized that surgery may avoid long-term or ineffective medical treatment improving quality of life and reducing costs. With the implementation of the laparoscopic approach, morbidity and overall costs are further reduced, and body image and cosmesis are maintained [5,11-15]. For these reasons time has come to compare laparoscopic ileocolic resection with infliximab treatment in terms of quality of life, sick leave and costs.Methods/designStudy objectivesTo compare, in a prospective randomized setting, the short-term and medium-term effectiveness and costs of ileocolic resection versus infliximab therapy in patients with recurrent Crohn's disease of the distal ileum. Two research questions can be defined:1. How does infliximab treatment of patients with recurrent Crohn's disease of the distal ileum compare with laparoscopic ileocolic resection in terms of quality of life, hospital stay, morbidity, sick leave and surgical (re)interventions?2. What are the 12 months cumulative total costs of infliximab treatment versus laparoscopic ileocolic resection in patients with recurrent Crohn's disease of the distal ileum?Study designThe LIR!C-study is a randomized multicenter trial with participation of at least five academic and 20 regional hospitals. Patients presenting with recurrent or resistant Crohn's disease of the distal ileum will be counseled and asked for informed consent if the inclusion and exclusion criteria are met. Randomization will take place after informed consent has been obtained. Patients will be randomized to either a laparoscopic ileocolic resection or to treatment with infliximab (see Figure 1).Figure 1LIR!C-trial flowchart.Randomization is performed by an Internet randomization module prepared by the Department of Clinical Research and Data management. Biased-coin randomization is used and the randomization is stratified for the randomizing centers and the presence or absence of peri-anal fistulas.Primary and secondary endpointsThe primary endpoints of the LIR!C trial are the disease-specific quality of life as measured by the IBDQ [16,17] and the costs per QALY.Secondary outcome parameters are general quality of life, as measured by the SF-36 [18] and EQ 5D [19,20] questionnaires; number of days on sick leave; morbidity (due to either surgery or medical treatment), total in and out hospital medical and non-medical costs and body image and cosmesis as measured by the body image questionnaire (BIQ) [13].Study populationThe study population consists of patients with recurrent or resistant Crohn's disease of the distal ileum, not responding to immunomodulating therapy.Inclusion criteria are: age in between 18 and 80 years, recurrent Crohn's disease of the distal ileum, a completed IBDQ, SF-36 and EQ-5D and BIQ before randomization, informed consent.Exclusion criteria are: prior ileocolic resection for Crohn's disease, obstructive Crohn's disease of the distal ileum requiring surgery, diseased small bowel segment longer than 40 cm, abdominal abscesses and abdominal fluid collections, American Society of Anesthesiologists (ASA) III en IV, insufficient understanding of the Dutch language or cognitively inability to complete Dutch questionnaires.Participating centersTwenty-seven centers will enroll patients. Five of these hospitals are academic hospitals.EthicsThe study is conducted in accordance with the principles of the Declaration of Helsinki and 'good clinical practice' guidelines. The protocol has been approved by the Medical Ethical Committee of the Academic Medical Center in Amsterdam and the local Ethical Committees of the participating centers. Prior to randomization informed consent will be obtained from patients.Study OutlineRecruitmentPatients will be recruited in the outpatients IBD clinics of the participating medical centers. Patients may not want to participate in the study because they reject the principle of randomization, especially since the randomization of this trial will result in two very divergent treatment strategies. A small pilot study in the AMC IBD clinic learned that most patients would participate as long as careful and clear explanation of the study is offered. In a prior study comparing laparoscopic with open ileocolic resection performed by our institute only 2 out of 62 patient refused participation or randomization [14].InfliximabPatients randomly allocated to infliximab treatment arm will undergo remission induction consisting of three subsequent infusions at week 0, 2 and 6 in a dose of 5 mg/kg. Infliximab maintenance therapy, consisting of infusions of 5 mg/kg at 8 to 12 weeks intervals, will be given to patients with active disease after an episode of disease activity that was treated with infliximab remission induction. In case of disease recurrence during infliximab treatment intervals will be shortened to 6 weeks and/or the dose level increased to 10 mg/kg. Infusion reactions will be treated with 25 mg prednisolone and 2 mg clemastine intravenously prior to subsequent infusions. Infliximab therapy will be combined with azathioprine immunomodulation in a dose of 2,5 mg/kg daily or 6-mercaptopurine 1,5 mg/kg daily. In the case of intolerance to these immunomodulating agents methotrexate will be given in an intramuscular dose of 15 mg once a week. Infliximab will be given without any co-medication in case of intolerance to abovementioned immunomodulating drugs or in case of contra-indications for the use of these drugs. Patients total blood counts and liver enzymes will be monitored 2 and 4 weeks after initiation of therapy and subsequently at 3 months intervals.(Laparoscopic) ileocolic resectionPatients randomly allocated to surgery receive a short course of steroids to reduce the inflammation, consisting of prednisolone 40 mg oral dose (OD) for one week, 30 mg OD during one week, 20 mg OD for 1 week, followed by a dose of 10 mg. Once steroid therapy has been tapered to a dose of 20 mg/day ileocolic resection can be performed.Surgery will be performed under general anesthesia. Patients will receive antibiotics for 24 hours. Ileocolic resection is done preferably laparoscopically. A variety of techniques can be applied performing a laparoscopic ileocolic resection ranging from a facilitated (laparoscopic mobilization of the right colon followed by extracorporeal vascular ligation, bowel transsection and reanastomosing) to a total laparoscopic procedure (all steps are done intracorporeally including anastomosis making). Generally, 3 or 4 trocarts suffice. The minilaparotomy preferably is done as an up and down transumbilical incision or in case of a large specimen as a Pfannenstiehl incision.Statistical analysisIntention to treatThe analysis will be performed in accordance with the intention to treat principle.Sample size calculationThe primary outcome of the study is a difference in IBDQ total score between the two randomized groups at one year. In order to assess the sample size for this study, a 0.5 between-group effect size on the IBDQ total score at week 48 was considered to be relevant. A modest effect size of 0.50 is generally considered to be clinically relevant. With a 5% two-sided significance level, 65 patients per study arm will be needed to achieve an 80% power to detect such a difference with a two-sided t-test [21]. Additional mixed-models repeated measures analysis of variance will be used to investigate whether there is a different pattern of change over time between the two study arms in the four IBDQ dimensions and the EQ 5D [22].Data collection and monitoringPatients will be followed for a period of 12 months. Seven times during this follow-up period patients will complete a set of questionnaires (the IBDQ, EQ 5D, SF-36 and BIQ): patients will complete the first set of questionnaires before randomization, the next set at week 2 of their therapy, the third set at week 6 and after that every 3 months (3, 6, 9 and 12 months after start of therapy). The questionnaires will be sent to the patients by post accompanied by a return envelop provided with postage stamps and the address of the hospital. Collection of the questionnaires will be safeguarded by the trial coordinator.Additional to the questionnaires, disease activity will be assessed by calculating the CDAI. For this calculation patients will be asked to keep a diary for seven days. In total, patients will fill in 7 diaries. During a visit to the gastro-enterologist or trial-nurse, hematocrit, presence or absence of an abdominal mass and number of complications will be assessed. CDAI can be calculated from these data combined with data from the CDAI-diaries. These visits will coincide with visits to the outpatient department for conventional patient care. At the end of the study period, after 12 months, patients will undergo an endoscopy to measure the extent of inflammation 12 months after therapy. Patients that received an ileocolic resection will be scored using the Rutgeerts endoscopic score.Patients will be contacted by telephone every month by a trial nurse to assess complications, additional interventions, re-admissions, duration of hospital and intensive care stay and visits to the outpatient clinic, number of days of sick leave and of social in attendance and to ensure completions of the questionnaires.An electronic Case Record Form (CRF) will include general patient's data (sex, age, medical history etc), patient's response to the questionnaires and data concerning type of intervention, complications, mortality, duration of hospital and intensive care stay.An independent trial monitor from the IBD trial-department will monitor the study procedure and the data of included patients.Data analysisAs we do not expect a difference in mortality, data on quality of life will be the key outcome measure in the comparison. Differences in quality of life and morbidity will be analyzed using mixed-models analysis of variance for repeated measures, accounting for differences in survival between groups. Mortality will be compared using Kaplan-Meier curves and log-rank statistics.To analyze the secondary outcomes (general quality of life, number of days on sick leave, morbidity (due to either surgery or medical treatment) and results of the body image questionnaire) the two groups will be compared using the statistical program SPSS 14.0®.A data and safety monitoring committee will safeguard trial continuation based on safety and effectiveness data. They will perform an interim analysis after 60 included patients have reached a one month follow up.Economic evaluationThe marginal direct medical, non-medical and time cost, costs per QALY and cost-utility ratio will be calculated for the surgical and medical treatment strategies. Cost items will include costs of hospital admissions and readmissions (operation, nursing days, outpatient visits), institutional care (nursing homes, hospice), home care, medication and other health care providers as well as direct non-medical costs (travel expenses). Costs will be calculated by counting resource use in the diaries, questionnaires and additional 3 month interviews and multiplying these with unit prices. Standard unit prices will be used when available, complemented by results from cost calculations where needed.The cumulative total costs will be calculated for the 12 month study period. In addition, the cumulative costs for each cost category will be calculated.The EQ-5D score profiles will be transposed to health utility values following scoring algorithms based on time trade-off elicitation techniques applied in the general population. Both the UK and the Dutch scoring algorithms will be applied and compared in sensitivity analyses. QALY's are calculated as the product-sum of health utilities and the lengths of the preceding period in-between measurements during follow-up. In the final analysis, a 12 month difference in average QALY's will be calculated.DiscussionIn the Netherlands infliximab treatment is indicated for patients with Crohn's disease that are either steroid refractory or steroid dependent following treatment with steroids alone or in combination with immunomodulatory drugs such as azathioprine or methotrexate [23]. These guidelines are in concert with the European consensus on infliximab treatment [4]. However, the ECCO also recommends in its consensus statement the need for trials comparing infliximab and surgery stating that 'infliximab should be considered for steroid or immunomodulatory disease or intolerance, although surgical options should be considered and discussed.' The LIR!C-trial aims to diminish this discussion and to provide an evidence-based best treatment strategy.Besides infliximab (Remicade®), new biologic agents have been introduced in the past few years. Adalimumab (Humira®), a human anti-TNF monoclonal antibody, has possible advantages over infliximab in administration route and costs. In the future, therefore, adalimumab might become the preferred biological treatment. Nevertheless, at this moment adalimumab as therapy for Crohn's disease is relatively young and still proving itself. Four placebo-controlled trials assessing efficacy in Crohn's disease have been conducted so far, analyzing 1400 patients. Conclusions were that adalimumab is more effective than placebo for remission-induction and maintaining remission [24-27]. No trials comparing adalimumab and infliximab head to head have been published yet. Because of lack of sufficient long-term data we chose to not use adalimumab in this study. Certoluzimab Pegol (Cimzia®), a polyethylene glycolated anti-TNFα antibody fragment was associated with a modest improvement in response rates but with no significant improvements in remission rates if compared to placebo [28]. As maintenance therapy outcomes on response and remission were better compared to placebo-therapy [29]. However, it has not been registered in Europe yet for the therapy of Crohn's disease and therefore it is not included in this trial either.This trial compares medical therapy with (minimal invasive) surgery for Crohn's disease of the distal ileum. The first analysis will provide short- and medium-term results up to one year of follow-up. However, since long-term data of this cohort are especially of importance, we aim to develop a follow-up study to continue follow-up after the first year.Considering the drawbacks of infliximab treatment, ileocolic resection can be an equivalent alternative treatment, in spite of a small risk on serious surgical complications. Both strategies have not been compared in a clinical trial so far [4]. Infliximab treatment may be less cost-effective when compared to laparoscopic ileocolic resection and may show less effective when assessed by means of quality of life. Therefore this study aims to answer the question which treatment is to be preferred for recurrent distal ileitis: medical therapy or early surgery.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsEJE drafted the manuscript. WAB and PCFS co-authored the writing of the manuscript. PCFS and WAB are the principal investigators. All other authors participated in the design of the study during several meetings and are local investigators at the participating centers. All authors edited the manuscript and read and approved the final manuscript.AcknowledgementsZonMw, grant number 10788.2201All authors are member of the LIR!C-study group.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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